EP2794653B1 - Konstante regionen gentechnisch hergestellter antikörper für stellenspezifische konjugation sowie verfahren und verwendungen dafür - Google Patents

Konstante regionen gentechnisch hergestellter antikörper für stellenspezifische konjugation sowie verfahren und verwendungen dafür Download PDF

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EP2794653B1
EP2794653B1 EP12823018.2A EP12823018A EP2794653B1 EP 2794653 B1 EP2794653 B1 EP 2794653B1 EP 12823018 A EP12823018 A EP 12823018A EP 2794653 B1 EP2794653 B1 EP 2794653B1
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engineered
polypeptide
antibody
amino acid
disclosure
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EP2794653A1 (de
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Kimberly MARQUETTE
Eric Bennett
Lioudmila Tchistiakova
L. Nathan Tumey
Jack Bikker
Valerie Calabro
Edmund Graziani
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Pfizer Inc
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Pfizer Inc
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the .txt file contains a sequence listing entitled "PC071868A_Sequence_Listing.txt" created on December 15, 2012, and having a size of 303 KB.
  • the sequence listing contained in this .txt file is part of the specification.
  • the present invention relates to antibodies, and fragments thereof, wherein at least one constant region is engineered to introduce an amino acid for site-specific conjugation.
  • the invention further relates to methods and uses of the engineered antibodides and fragments for, among other things, production of antibody-drug conjugate therapeutics.
  • Cancer is the second leading cause of death in the United States with one in two men and one in three women diagnosed with cancer at some time during their lifetime.
  • chemotherapeutic agents Although many chemotherapeutic agents have been developed, they often demonstrate unacceptable toxicity and or lack of specificity for cancer cells over non-cancer tissues. To avoid the non-specific cytotoxic effects of chemotherapeutic agents, targeted antibody therapy has revolutionized cancer treatment, with several monoclonal antibodies (mAbs) demonstrating clinical potential. Because antibodies against tumor-specific antigens often lack therapeutic activities, they have been conjugated to cytotoxic agents in order to combine the effectiveness of chemotherapy with the targeting of antibodies. In principle, selective delivery of cytotoxic agents to specific tumor issues by antibody binding should reduce the systemic toxicity of traditional small-molecule chemotherapeutics.
  • mAbs monoclonal antibodies
  • Antibodies have been conjugated to a variety of cytotoxic drugs, including small molecules that alkylate DNA (e.g., duocarmycin and calicheamicin), disrupt microtubules (e.g., maytansinoids and auristatins) or bind DNA (e.g., anthracyclins).
  • ADC antibody-drug conjugate
  • calicheamicin - MylotargTM a humanized anti-CD33 antibody conjugated to calicheamicin - MylotargTM (gemtuzumab ozogamicin, Wyeth) - was approved in 2000 for acute myeloid leukemia.
  • AdcetrisTM (brentuximab vedotin; Seattle Genetics), an ADC comprising a chimeric antibody to CD30 conjugated to the auristatin monomethyl auristatin E (MMAE; also referred to as N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine) for treatment of Hodgkin's lymphoma and anaplastic large cell lymphoma.
  • MMAE auristatin monomethyl auristatin E
  • ADCs hold promise for cancer therapy
  • cytotoxic drugs are generally conjugated to the antibodides via lysine side chains or by reducing interchain disulfide bonds present in the antibodies to provide activated cysteine sulfhydryl groups.
  • This non-specific conjugation approach has numerous drawbacks. Not only is it capable of affecting protein folding by disrupting cystine bonds, non-specific conjugation creates a heterogeneous mixture of antibodies having a diverse mix of antibody-to-drug ratios (ADR) and also having a complex mixture of antibodies conjugated at a variety of positions. So, even if it was somehow possible to purify sufficient antibodies having a desired antibody:drug ratio, the fraction would still comprise a complex mix of antibodies conjugated at various positions. Each species could potentially have distinct therapeutic properties, and batch-to-batch consistency would be difficult to control, all of which present significant hurdles to success of using ADC for cancer therapy.
  • WO 2011/005481 biotin-maleimide conjugation
  • WO 2010/141902 conjugating cysteine variants with maleimide dyes
  • WO 2006/034488 biotin-maleimide conjugation was performed and all examples describing conjugation to monomethyl auristatin E (MMAE; N-methylvaline-valine-dolaisoleucine-dolaproine-norephedrine) and monomethyl auristatin F (MMAF; also referred to as "N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine" were prophetic only).
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine monomethyl auristatin F
  • WO2006034488 discloses antibodies engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids.
  • the invention relates to an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index numbering system as set forth in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 .
  • C ⁇ constant domain
  • the invention may further comprise at least one further amino acid substitution selected from the group consisting of D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, N421, M428, A431, L432, T437, Q438, K439, L443, and S444, according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 .
  • the invention may further comprise at least one mutation selected from the group consisting of a mutation at amino acid position 284, 287, A327, N384, L398, and V422, according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 .
  • the invention may comprise SEQ ID NO:108.
  • the engineered C ⁇ polypeptide may comprise SEQ ID NO:6.
  • the engineered polypeptide of the invention may be conjugated to one or more of a cytotoxic agent, cytostatic agent, chemotherapeutic agent, toxin, radionuclide, DNA, RNA, siRNA, microRNA, peptide nucleic acid, non-natural amino acid, peptide, enzyme, fluorescent tag, and biotin, wherein the conjugation is at the substituted cysteine, and wherein the cytotoxic agent is optionally conjugated to the polypeptide via a linker, and the linker may be selected from the group consisting of mc (maleimidocaproyl), val-cit (valine-citrulline), mc-val-cit (maleimidocaproyl-valine-citrulline), mc-val-cit-PABC (maleimidocaproyl-valine-citrulline- p -aminobenzylcarbamate), Mal-PEG2C2 (maleimido-[CH 2 CH 2 O] 2 CH 2 CH
  • the cytotoxic agent may be selected from the group consisting of an auristatin, a maytansinoid and a calicheamicin.
  • the linker and the cytotoxic agent may be selected from the group consisting of maleimidocaproyl-monomethyl auristatin D (mcMMAD), maleimidocaproyl-0101 (mc0101), maleimidocaproyl-3377 (mc3377), maleimidocaproyl-8261 (mc8261), valine-citrulline-monomethyl auristatin D (vcMMAD), valine-citrulline-0101 (vc0101), valine-citrulline-3377 (vc3377), valine-citrulline-8261 (vc8261), mcValCitPABCMMAD (maleimidocaproyl-valine-citrulline-monomethyl auristatin D), mcValCit0101 (maleimidocaproyl-valine
  • the invention comprises an Fc fusion protein comprising the engineered C ⁇ polypeptide of the invention.
  • the invention comprises an antibody, or antigen-binding portion thereof, comprising the engineered C ⁇ polypeptide of the invention, and further comprising a light chain comprising an engineered constant domain selected from the group consisting of:
  • the invention comprises a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered C ⁇ polypeptide of the invention, or a portion thereof, or an antibody of the invention or antigen-binding portion thereof, and a pharmaceutically acceptable carrier.
  • the compositions of the invention may be for use in a method of treating cancer, autoimmune, inflammatory, or infectious diseases or disorders in a subject in need thereof.
  • the invention further provides a nucleic acid encoding the engineered C ⁇ polypeptide of the invention, or a portion thereof, or the antibody of the invention or antigen-binding portion thereof.
  • the invention also provides a host cell comprising such a nucleic acid.
  • the invention provides a method of producing an engineered C ⁇ polypeptide of the invention, or a portion thereof, or an antibody of the invention or antigen-binding portion thereof, comprising incubating a host cell of the invention under suitable conditions for expressing the engineered C ⁇ polypeptide, or portion thereof, or the antibody, or antigen-binding portion thereof, and isolating the engineered C ⁇ polypeptide, or portion thereof, or the antibody or antigen-binding portion thereof.
  • the disclosure includes an engineered antibody constant domain polypeptide, or a portion thereof, wherein the engineered constant domain comprises at least one amino acid substitution to introduce a cysteine residue useful for conjugation, wherein the constant domain polypeptide is:
  • the engineered C ⁇ polypeptide further comprises at least one mutation selected from the group consisting of a mutation at amino acid position 284, 287, A327, N384, L398, and V422, according to the EU index of Kabat.
  • the engineered C ⁇ polypeptide comprises one or more of the following pairs of amino acid substitutions: a) E380 and L443; b) L398 and L443; c) V422 and L443; d) E380 and L398; e) L398 and V422; f) E380 and V422; g) K392 and L443; h) F404 and L443; and i) K392 and F404.
  • the disclosure also provides engineered C ⁇ polypeptide comprising an amino acid sequence selected from the group consisting of (a) the amino acid sequence of SEQ ID NO:99 and the amino acid sequence of SEQ ID NO:107; (b) the amino acid sequence of SEQ ID NO:103 and the amino acid sequence of SEQ ID NO:107; (c) the amino acid sequence of SEQ ID NO:105 and the amino acid sequence of SEQ ID NO:107; (d) the amino acid sequence of SEQ ID NO:99 and the amino acid sequence of SEQ ID NO:103; (e) the amino acid sequence of SEQ ID NO:103 and the amino acid sequence of SEQ ID NO:105; (f) the amino acid sequence of SEQ ID NO:99 and the amino acid sequence of SEQ ID NO:105; (g) the amino acid sequence of SEQ ID NO:102 and the amino acid sequence of SEQ ID NO:107; (h) the amino acid sequence of SEQ ID NO:104 and the amino acid sequence of SEQ ID NO:107; and (i) the amino acid sequence of SEQ ID NO:
  • the engineered C ⁇ polypeptide is selected from an IgG1, IgG2, IgG3, or an IgG4 subclass.
  • the engineered antibody constant domain polypeptide, or a portion thereof is conjugated to one or more of a cytotoxic agent, cytostatic agent, chemotherapeutic agent, toxin, radionuclide, DNA, RNA, siRNA, microRNA, peptide nucleic acid, non-natural amino acid, peptide, enzyme, fluorescent tag, and biotin, wherein the conjugation is at the substituted cysteine.
  • the cytotoxic agent is conjugated to the polypeptide via a linker.
  • mc maleimidocaproyl
  • val-cit valine-citrulline
  • mc-val-cit-PABC maleimidocaproyl-valine-citrulline-p-a
  • the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid and a calicheamicin.
  • the linker and the cytotoxic agent are selected from the group consisting of maleimidocaproyl-monomethyl auristatin D (mcMMAD), maleimidocaproyl-0101 (mc0101), maleimidocaproyl-3377 (mc3377), maleimidocaproyl-8261 (mc8261), valine-citrulline-monomethyl auristatin D (vcMMAD), valine-citrulline-0101 (vc0101), valine-citrulline-3377 (vc3377), valine-citrulline-8261 (vc8261), mcValCitPABCMMAD (maleimidocaproyl-valine-citrulline-monomethyl auristatin D), mcValCit0101 (maleimidocaproyl-valine-citrulline-0101), mcValCit3377 (maleimidocaproyl-valine-citrulline-3377), mcValC
  • the invention further comprises an antibody, or antigen-binding portion thereof, comprising an engineered C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, N421, M428, A431, L432, T437, Q438, K439, L443, and S444, according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 .
  • the antibody, or antigen-binding portion thereof further comprises an engineered human lambda light chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K110, A111, L125, K149C, V155, G158, T161, Q185, S188, H189, S191, T197, V205, E206, K207, T208 and A210, according to the numbering of Kabat.
  • C ⁇ human lambda light chain constant domain
  • the antibody, or antigen-binding portion thereof further comprises an engineered human kappa light chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of A111, K183, and N210, according to the numbering of Kabat.
  • C ⁇ constant domain
  • the antibody, or antigen-binding portion thereof further comprises an engineered C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, N421, M428, A431, L432, T437, Q438, K439, L443, and S444, according to the EU index of Edelman et al., 1969, Proc.
  • the antibody further comprises a C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K110, A111, L125, K149C, V155, G158, T161, Q185, S188, H189, S191, T197, V205, E206, K207, T208 and A210, according to the numbering of Kabat, and further comprises a C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of A111, K183, and N210, according to the numbering of Kabat.
  • the invention further comprises an antibody, or antigen-binding portion thereof, comprising an engineered C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K110, A111, L125, K149C, V155, G158, T161, Q185, S188, H189, S191, T197, V205, E206, K207, T208 and A210, according to the numbering of Kabat.
  • the invention further comprises an an antibody, or antigen-binding portion thereof, comprising an engineered C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of A111, K183, and N210, according to the numbering of Kabat.
  • the invention provides an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index numbering system according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 and wherein the constant domain polypeptide is at least one of:
  • the invention provides an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci.
  • C ⁇ constant domain
  • USA 63(1):78-85 and further comprises an engineered heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, N421, M428, A431, L432, T437, Q438, K439, L443, and S444, and further comprising a light chain comprising an engineered constant domain selected from the group consisting of:
  • the antibody, or antigen-binding portion thereof further comprises at least one of:
  • the invention includes an Fc fusion protein comprising an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci.
  • C ⁇ constant domain
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody, or antigen-binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen-binding portion thereof, comprises an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 , and optionally further comprising
  • C ⁇ human IgG heavy chain constant domain
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody, or antigen-binding portion thereof, and a pharmaceutically acceptable carrier, wherein the antibody, or antigen-binding portion thereof, comprises an engineered heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K246, D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, N315, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, Q386, E388, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, Q419, N421, M428, A431, L432, T437, Q43
  • the invention includes a method of treating cancer, autoimmune, inflammatory, or infectious diseases or disorders in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, or an Fc fusion protein, wherein the antibody, or antigen-binding portion thereof, or the Fc fusion protein, comprises an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 , and wherein the engineered constant domain polypeptide optionally further comprises:
  • the engineered constant domain polypeptide is a C ⁇ polypeptide further comprising at least one mutation selected from the group consisting of a mutation at amino acid position 284, 287, 327, 359, 361, 383, 384, 398, and 422, according to the EU index of Kabat.
  • the antibody, or antigen-binding portion thereof comprises an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K246, D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, N315, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, Q386, E388, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, Q419, N421, M428, A431, L432, T437, Q438, K439, L443, and S444, according to the EU index of Kabat, and further comprises at least
  • the engineered constant domain polypeptide, or portion thereof is conjugated to one or more of a cytotoxic agent, cytostatic agent, chemotherapeutic agent, toxin, radionuclide, DNA, RNA, siRNA, microRNA, peptide nucleic acid, non-natural amino acid, peptide, enzyme, fluorescent tag, and biotin, and wherein the conjugation is at the substituted amino acid.
  • the antibody comprises an engineered constant domain polypeptide, or portion thereof, and further comprises a linker and a cytotoxic angent, wherein the linker and the cytotoxic agent are selected from the group consisting of maleimidocaproyl-monomethyl auristatin D (mcMMAD), maleimidocaproyl-0101 (mc0101), maleimidocaproyl-3377 (mc3377), maleimidocaproyl-8261 (mc8261), valine-citrulline-monomethyl auristatin D (vcMMAD), valine-citrulline-0101 (vc0101), valine-citrulline-3377 (vc3377), valine-citrulline-8261 (vc8261), mcValCitPABCMMAD (maleimidocaproyl-valine-citrulline-monomethyl auristatin D), mcValCit0101 (maleimidocaproyl-va
  • the invention includes a nucleic acid encoding an engineered constant domain polypeptide of or a portion thereof, wherein the engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 , and optionally further comprising
  • the invention includes a nucleic acid encoding an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at least one substitution being located at K246 according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci.
  • C ⁇ constant domain
  • the invention includes a host cell comprising the nucleic acid encoding the engineered Fc polypeptide.
  • the invention includes a nucleic acid encoding an engineered C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of A111, K183, and N210, according to the numbering of Kabat;
  • the invention includes a host cell comprising the nucleic acid encoding the engineered C ⁇ polypeptide, or portion thereof.
  • the invention includes a nucleic acid encoding the engineered C ⁇ polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K110, A111, L125, K149C, V155, G158, T161, Q185, S188, H189, S191, T197, V205, E206, K207, T208 and A210, according to the numbering of Kabat;
  • the invention includes a nucleic acid encoding an engineered human IgG heavy chain constant domain (C ⁇ ) polypeptide, or portion thereof, comprising at least one amino acid substitution to introduce a cysteine residue useful for conjugation, said at
  • the antibody comprises at least one engineered antibody constant domain polypeptide, or a portion thereof, wherein the engineered constant domain polypeptide comprises at least one amino acid substitution to introduce a cysteine residue useful for conjugation, and wherein the engineered constant domain polypeptide is selected from the group consisting of:
  • the invention comprises a host cell comprising the nucleic acid.
  • the invention includes a method of producing an engineered antibody, or antigen-binding portion thereof, comprising incubating the host cell under suitable conditions for expressing the antibody, or antigen-binding portion thereof, and isolating the antibody or antigen-binding portion.
  • Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein.
  • the nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • an element means one element or more than one element.
  • references to "about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X” includes a description of "X.” Numeric values are inclusive of numbers defining the range.
  • the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members.
  • the present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
  • amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table: Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D Glutamic Acid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr Y Cysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S Threonine Thr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan Trp W
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity).
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994 ).
  • Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-45 (1992 ).
  • a "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, and (4) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs comprising substitutions, deletions, and/or insertions can include various muteins of a sequence other than the specified peptide sequence.
  • single or multiple amino acid substitutions may be made in the specified sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts, e.g., outside of the CDRs).
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984 )); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991 )); and Thornton et al., Nature 354:105 (1991 ).
  • Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • Genetics Computer Group (GCG available from Genetics Computer Group, Inc.), also referred to as the Wisconsin Package, is an integrated software package of over 130 programs for accessing, analyzing and manipulating nucleotide and protein sequences. GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters to determine sequence similarity, homology and/or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG version 6.1, version 7.0, version 9.1, and version 10.0.
  • Polypeptide sequences also can be compared using FASTA, a program in GCG, using default or recommended parameters.
  • FASTA e.g., FASTA2 and FASTA3
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences ( Pearson, Methods Enzymol. 183:63-98 (1990 ); Pearson, Methods Mol. Biol. 132:185-219 (2000 )).
  • Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters. See, e.g., Altschul et al., J. Mol. Biol. 215:403-410 (1990 ); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997 ).
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • upstream refers to a residue that is N-terminal to a second residue where the molecule is a protein, or 5' to a second residue where the molecule is a nucleic acid.
  • downstream refers to a residue that is C-terminal to a second residue where the molecule is a protein, or 3' to a second residue where the molecule is a nucleic acid.
  • nucleic acid is a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term is used to include single-stranded nucleic acids, double-stranded nucleic acids, and RNA and DNA made from nucleotide or nucleoside analogues.
  • vector refers to a nucleic acid molecule that may be used to transport a second nucleic acid molecule into a cell.
  • the vector allows for replication of DNA sequences inserted into the vector.
  • the vector may comprise a promoter to enhance expression of the nucleic acid molecule in at least some host cells.
  • Vectors may replicate autonomously (extrachromasomal) or may be integrated into a host cell chromosome.
  • the vector may comprise an expression vector capable of producing a protein derived from at least part of a nucleic acid sequence inserted into the vector.
  • hybridization may be to filter bound DNA using hybridization solutions standard in the art such as 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), at 65°C, and washing in 0.25 M NaHPO 4 , 3.5% SDS followed by washing 0.1 x SSC/0.1% SDS at a temperature ranging from room temperature to 68°C depending on the length of the probe (see e.g. Ausubel, F.M.
  • a high stringency wash comprises washing in 6x SSC/0.05% sodium pyrophosphate at 37°C for a 14 base oligonucleotide probe, or at 48°C for a 17 base oligonucleotide probe, or at 55°C for a 20 base oligonucleotide probe, or at 60°C for a 25 base oligonucleotide probe, or at 65°C for a nucleotide probe about 250 nucleotides in length.
  • Nucleic acid probes may be labeled with radionucleotides by end-labeling with, for example, [ ⁇ - 32 P]ATP, or incorporation of radiolabeled nucleotides such as [ ⁇ - 32 P]dCTP by random primer labeling.
  • probes may be labeled by incorporation of biotinylated or fluorescein labeled nucleotides, and the probe detected using Streptavidin or anti-fluorescein antibodies.
  • fusion protein refers to a protein or polypeptide that has an amino acid sequence derived from two or more proteins.
  • the fusion protein may also include linking regions of amino acids between amino acid portions derived from separate proteins.
  • host cell refers to a cell that is grown in culture according to the present invention to produce a protein or polypeptide of interest.
  • the host cell is a mammalian cell.
  • hybridoma is meant to encompass a cell or progeny of a cell resulting from fusion of an immortalized cell and an antibody-producing cell.
  • the resulting hybridoma is an immortalized cell that produces antibodies.
  • the individual cells used to create the hybridoma can be from any mammalian source, including, but not limited to, rat, pig, rabbit, sheep, goat, and human.
  • the term also encompasses trioma cell lines, which result when progeny of heterohybrid myeloma fusions, which are the product of a fusion between human cells and a murine myeloma cell line, are subsequently fused with a plasma cell.
  • the term is meant to include any immortalized hybrid cell line that produces antibodies such as, for example, quadromas (See, e.g., Milstein et al., 1983, Nature 537:3053 ).
  • polypeptide refers a sequential chain of amino acids linked together via peptide bonds.
  • the term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond.
  • polypeptides may be processed and/or modified.
  • a polypeptide may be glycosylated.
  • a polypeptide to be expressed according to the present invention can be a therapeutic polypeptide.
  • a therapeutic polypeptide is a polypeptide that has a biological effect on a region in the body on which it acts or on a region of the body on which it remotely acts via intermediates. Examples of therapeutic polypeptides are discussed in more detail below.
  • Protein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms "polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of multiple polypeptides that physically associate with one another, the term “protein” as used herein refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.
  • a protein to be expressed according to the present invention can be a protein therapeutic.
  • a protein therapeutic is a protein that has a biological effect on a region in the body on which it acts or on a region of the body on which it remotely acts via intermediates. Examples of protein therapeutics are discussed in more detail below.
  • fragment refers to a polypeptide and is defined as any discrete portion of a given polypeptide that is unique to or characteristic of that polypeptide.
  • the term as used herein also refers to any discrete portion of a given polypeptide that retains at least a fraction of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the activity of the full-length polypeptide.
  • the fraction of activity retained is at least 95%, 96%, 97%, 98% or 99% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity retained is 100% or more of the activity of the full-length polypeptide.
  • the term as used herein also refers to any portion of a given polypeptide that includes at least an established sequence element found in the full-length polypeptide. In some embodiments, the sequence element spans at least about 4-5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the full-length polypeptide.
  • Recombinantly expressed polypeptide and “recombinant polypeptide” as used herein refer to a polypeptide expressed from a host cell that has been manipulated to express that polypeptide.
  • the host cell is a mammalian cell.
  • this manipulation may comprise one or more genetic modifications.
  • the host cells may be genetically modified by the introduction of one or more heterologous genes encoding the polypeptide to be expressed.
  • the heterologous recombinantly expressed polypeptide can be identical or similar to polypeptides that are normally expressed in the host cell.
  • the heterologous recombinantly expressed polypeptide can also be foreign to the host cell, e.g. heterologous to polypeptides normally expressed in the host cell.
  • the heterologous recombinantly expressed polypeptide is chimeric.
  • portions of a polypeptide may contain amino acid sequences that are identical or similar to polypeptides normally expressed in the host cell, while other portions contain amino acid sequences that are foreign to the host cell.
  • a polypeptide may contain amino acid sequences from two or more different polypeptides that are both normally expressed in the host cell.
  • a polypeptide may contain amino acid sequences from two or more polypeptides that are both foreign to the host cell.
  • the host cell is genetically modified by the activation or upregulation of one or more endogenous genes.
  • An intact "antibody” comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989 )).
  • Each heavy chain is comprised of a heavy chain variable region (HCVR or V H ) and a heavy chain constant region (C H ).
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (LCVR or V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989 )).
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a tumor-associated antigen, TAA). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • an antigen e.g., a tumor-associated antigen, TAA
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H 1 domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment ( Ward et al., (1989) Nature 341:544-546 ), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR), disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies and intrabodies.
  • CDR complementarity determining region
  • dsFv
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv)); see e.g., Bird et al. Science 242:423-426 (1988 ) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988 )).
  • scFv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which V H and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993 ); Poljak et al., 1994, Structure 2:1121-1123 ).
  • Antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g. chickens), fish (e.g., sharks) and camelids (e.g., llamas).
  • IgG Fc region refers to the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule.
  • the terms relate to the constant region of an antibody excluding the first constant region immunoglobulin domain and further relates to portions of that region.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains, or portions thereof.
  • Fc may include the J chain.
  • Fc comprises immunoglobulin domains C ⁇ 2 and C ⁇ 3 (C gamma 2 and C gamma 3) and the hinge between C ⁇ 1 (C gamma 1) and C ⁇ 2 (C gamma 2).
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index of Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 as described in Kabat et al., 1991.
  • the Fc domain comprises from about amino acid residue 236 to about 447 of the human IgG1 constant domain.
  • Fc polypeptide may refer to this region in isolation, or this region in the context of an antibody, or an antigen-binding portion thereof, or Fc fusion protein.
  • engineered Fc polypeptides which are non-naturally occurring variants of an Fc comprising at least one amino acid substitution introducing a site-specific conjugation site.
  • the heavy chain constant domain comprises the Fc region and further comprises the CH1 domain and hinge as well as the CH2 and CH3 (and, optionally, CH4 of IgA and IgE) domains of the IgG heavy chain.
  • An exemplary human wild type IgG1 constant domain amino acid sequence is set forth in SEQ ID NO:1 and is shown in Figure 15A .
  • engineered Fc polypeptide By “engineered Fc polypeptide”, “engineered Fc region” and “engineered Fc” as the terms are interchangeably used herein, is meant an Fc polypeptide, or portion thereof, comprising at least one mutation, e.g., an amino acid substitution, introducing a site for conjugation.
  • the mutation introduces a cysteine in place of the naturally-occurring amino acid residue at that position, where the mutation creates a reactive site (e.g., a reactive sulfhydryl group) for conjugation of a moiety to the Fc.
  • engineered Fc variant refers to an engineered Fc polypeptide further comprising at least one additional modification, such as, but not limited to, an amino acid mutation, a post-translational modification (e.g., altered glycosylation), among others, in addition to the mutation creating a conjugation site.
  • additional modification such as, but not limited to, an amino acid mutation, a post-translational modification (e.g., altered glycosylation), among others, in addition to the mutation creating a conjugation site.
  • Hinge region is generally defined as stretching from Glu216 to Pro230 of human IgG1 ( Burton, 1985, Molec. Immunol. 22: 161-206 ), and refers to the portion of an IgG molecule comprising the C-terminal portion of the CH1 domain and the N-terminal portion of the CH2 domain.
  • Exemplary hinge regions for human IgG1, IgG2, IgG2 and IgG4 and mouse IgG1 and IgG2A are provided in US Patent No. 6,165,476 , at the Table shown at column 4, line 54 to column 5, line 15, and also illustrated, for example, in Janeway et al., 1999, Immunology: The Immune System in Health and Disease, 4th ed.
  • Hinge regions of other IgG isotypes may be aligned with the IgG 1 sequence by placing the first and last cysteine residues forming inter-heavy chain S--S bonds in the same positions.
  • An exemplary alignment of the constant domains of human IgG1, IgG2, IgG3, and IgG4 showing the alignment of the hinge region of each subclass is shown in Figure 19B .
  • the "lower hinge region" of an Fc region is normally defined as the stretch of residues immediately C-terminal to the hinge region, i.e. residues 233 to 239 of the Fc region.
  • IgG hinge-Fc region or "hinge-Fc fragment” as used herein refers to a hinge region (approximately residues 216-230) and an Fc region (residues 231-447) C-terminal thereto.
  • an “engineered Kappa light chain” as the term is used herein, refers to a Kappa light chain, or a portion thereof, comprising an engineered Kappa light chain constant region (C ⁇ ) comprising at least one amino acid substitution to introduce a reactive group useful for conjugation at that site.
  • Engineered kappa constant region mean the constant region of a kappa light chain, or a portion thereof, comprising at least one amino acid mutation to introduce an amino acid comprising a reactive group useful for conjugation compared with a wild type kappa constant region that is not so modified.
  • An exemplary human wild type kappa constant region amino acid sequence is as shown in Figure 18A and set forth in SEQ ID NO:89.
  • Engineerered lambda constant region mean the constant region of a lambda light chain, or a portion thereof, comprising at least one amino acid mutation to introduce an amino acid comprising a reactive group useful for conjugation compared with a wild type kappa constant region that is not so modified.
  • engineered antibody means an antibody, or antigen binding portion thereof, comprising at least one engineered constant region, e.g., an engineered Fc region, an engineered C ⁇ region and/or an engineered C ⁇ region.
  • engineered antibody antigen-binding portion or “engineered antibody portion,” as used herein, is meant an antigen-binding fragment of an antibody, e.g., a Fab, a F(ab') 2 , and the like, comprising at least one engineered constant region.
  • an antibody or antigen-binding portion thereof may be part of larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule ( Kipriyanov et al. Human Antibodies and Hybridomas 6:93-101 (1995 )) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules ( Kipriyanov et al. Mol. Immunol. 31:1047-1058 (1994 )).
  • CDRs from an antibody are incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin that specifically binds to an antigen of interest, such as a tumor antigen.
  • the CDR(s) may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated noncovalently.
  • Antibody portions, such as Fab and F(ab') 2 fragments can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • an antigen-binding portion thereof may also be used.
  • An antigen-binding portion competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989 )).
  • Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antigen-binding portions include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, single chain antibodies such as those derived from camelids or shark immunoglobulin novel antigen receptors (IgNARs), and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
  • the binding sites may be identical to one another or may be different.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope
  • human antibody or “fully human antibody”, as used herein, are intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure or antigen binding portions thereof may include amino acid residues not encoded by human germline immunoglobulin sequences ( e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo ).
  • the term "human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human monoclonal antibody or “fully human monoclonal antibody” refer to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, where the B cell is fused to an immortalized cell.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • isotype or “class” refers to the antibody class (e.g ., IgM or IgG) that is encoded by the heavy chain constant region genes.
  • the constant domains of antibodies are not involved in binding to antigen, but exhibit various effector functions.
  • a given human antibody or immunoglobulin can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM.
  • the structures and three-dimensional configurations of different classes of immunoglobulins are well-known. Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. Human IgG1 and IgG3 are known to mediate ADCC in humans.
  • subclass refers to the further specification within an isotype of the heavy chain constant region gene, such as, for example, the IgG1, IgG2, IgG3, or IgG4 subclasses within the IgG isotype.
  • an antibody recognizing an antigen and "an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”
  • ADCC antibody dependent cellular cytotoxicity
  • cytotoxic cells e.g. NK cells, neutrophils, macrophages, etc.
  • cytotoxic cells that mediate ADCC generally express Fc receptors (FcR).
  • the primary cells for mediating ADCC NK cells
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, and/or Fc ⁇ RIV.
  • FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991 ).
  • ADCC activity of a molecule may be assessed in vitro, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecules of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-656 .
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody where the Fc region comprises a hinge region and the C H 2 and C H 3 domains of the heavy chain.
  • the FcR can be a native sequence human FcR.
  • the FcR can be one that binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, and Fc ⁇ RIV subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, Daeron, 1997, Annu. Rev. Immunol. 15:203-234 ).
  • FcRs are reviewed in Ravetch and Kinet, 1991, Annu. Rev. Immunol.
  • FcR neonatal receptor
  • the primary FcR binding site on immunoglobulin Fc fragments resides in the hinge region between the C H 1 and C H 2. This hinge region interacts with the FcR1-3 on various leukocytes and trigger these cells to attack the target. ( Wines et al., 2000, J. Immunol. 164:5313-5318 ).
  • the hinge region encompasses, but is not limited to, the sequences described in U.S. Patent No. 6,165,476 .
  • ADCC antibody dependent cellular cytotoxicity
  • human antibody derivatives refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody.
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • a compound e.g., a protein, a nucleic acid, an antibody, and the like, which recognizes and binds a specific molecule, but does not substantially recognize or bind other molecules in a sample.
  • an antibody or a peptide inhibitor which recognizes and binds a cognate ligand (e.g ., an anti-IgE antibody that binds with its cognate antigen, IgE) in a sample, but does not substantially recognize or bind other molecules in the sample.
  • the specified binding moiety binds preferentially to a particular target molecule, e.g., IgE, and does not bind in a significant amount to other components present in a test sample.
  • a variety of assay formats may be used to select an antibody that specifically binds a molecule of interest. For example, solid-phase ELISA immunoassay, immunoprecipitation, BIAcore, FACS, and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with IgE.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background, even more specifically, an antibody is said to "specifically bind" an antigen when the equilibrium dissociation constant (K D ) is ⁇ 1 ⁇ M, preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • K D equilibrium dissociation constant
  • binding affinity is herein used as a measure of the strength of a non-covalent interaction between two molecules, e.g., and antibody, or fragment thereof, and an antigen.
  • binding affinity is used to describe monovalent interactions (intrinsic activity).
  • Binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determination of the dissociation constant (K D ).
  • K D can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method (Biacore).
  • the rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants ka (or k on ) and dissociation rate constant k d (or k off ), respectively.
  • binding affinities associated with different molecular interactions e.g. comparison of the binding affinity of different antibodies for a given antigen, may be compared by comparison of the K D values for the individual antibody/antigen complexes.
  • the specificity of an interaction may be assessed by determination and comparison of the K D value for the interaction of interest, e.g. a specific interaction between an antibody and an antigen, with the K D value of an interaction not of interest.
  • K on is intended to refer to the on-rate, or association rate of a particular antibody-antigen or receptor-ligand interaction
  • k off is intended to refer to the off-rate, or dissociation rate of a particular antibody-antigen/receptor-ligand interaction
  • K D is intended to refer to the dissociation constant, which is obtained from the ratio of k off to k on ( i.e,. k off /k on ) and is expressed as a molar concentration (M).
  • K D values for antibodies or other binding partners can be determined using methods well established in the art. One method for determining the K D is by using surface plasmon resonance, typically using a biosensor system such as a Biacore® system.
  • chimeric antibody as used herein means an antibody that comprises regions from two or more different antibodies.
  • a "chimeric antibody” comprises variable region sequences derived from one species and constant region sequences derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • one or more of the CDRs are derived from a mouse anti-human tumor antigen antibody.
  • all of the CDRs are derived from a mouse anti-human tumor antigen antibody.
  • the CDRs from more than one mouse anti-human tumor antigen antibodies are combined in a chimeric human antibody.
  • a chimeric antibody may comprise a CDR1 from the light chain of a first mouse anti-human tumor antigen antibody, a CDR2 from the light chain of a second mouse anti-human tumor antigen antibody and a CDR3 and CDR3 from the light chain of a third mouse anti-human tumor antigen antibody, and the CDRs from the heavy chain may be derived from one or more other anti-human tumor antigen antibodies.
  • the framework regions may be derived from one of the same mouse anti-human tumor antigen antibodies or from one or more different mice.
  • chimeric antibody includes an antibody comprising a portion derived from the germline sequences of more than one species.
  • glycoform refers to a complex oligosaccharide structure comprising linkages of various carbohydrate units. Such structures are described in, e.g., Essentials of Glycobiology Varki et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999 ), which also provides a review of standard glycobiology nomenclature. Such glycoforms include, but are not limited to, G2, G1, G0, G-1, and G-2 ( see, e.g., International Patent Publication No. WO 99/22764 ).
  • glycosylation pattern is defined as the pattern of carbohydrate units that are covalently attached to a protein (e.g., the glycoform) as well as to the site(s) to which the glycoform(s) are covalently attached to the peptide backbone of a protein, more specifically to an immunoglobulin protein.
  • Antibody-drug conjugate refers to an antibody, or a portion of an antibody, covalently linked to a cytotoxic or cytostatic drug/agent where the drug/agent is also referred to herein as a "payload.”
  • the antibody and the drug may be directly linked or they may be linked via a moiety referred to as a "linker.”
  • an effective amount is meant an amount that when administered to a mammal, preferably a human, mediates a detectable therapeutic response compared to the response detected in the absence of the compound.
  • a therapeutic response such as, but not limited to, inhibition of and/or decreased tumor growth, tumor size, metastasis, and the like, can be readily assessed by a plethora of art-recognized methods, including, e.g., such methods as disclosed herein.
  • the effective amount of the compound or composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the stage of the disease, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, the level of expression/availability of the target of the antibody-drug-conjugate, and the like.
  • Compet By the term “compete”, as used herein with regard to an antibody, is meant that a first antibody, or an antigen-binding portion thereof, competes for binding with a second antibody, or an antigen-binding portion thereof, where binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody.
  • the alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope.
  • each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitope(s).
  • cross-competing antibodies can bind to the epitope, or potion of the epitope, to which the antibodies used in the invention bind. Use of both competing and cross-competing antibodies is encompassed by the present invention.
  • epitope includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compound, combination, and/or composition of the invention in the kit for affecting, alleviating or treating the various diseases or disorders recited herein.
  • the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell, a tissue, or a mammal, including as disclosed elsewhere herein.
  • the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition.
  • the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.
  • patient refers to a human.
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • telomere binding partner e.g., an anti-human tumor antigen antibody that binds a tumor antigen
  • the specified binding moiety e.g., an antibody or an antigen-binding portion thereof or a receptor or a ligand binding portion thereof
  • a variety of assay formats may be used to select an antibody or peptide that specifically binds a molecule of interest.
  • solid-phase ELISA immunoassay immunoprecipitation
  • immunoprecipitation BIAcoreTM (GE Healthcare, Piscataway, NJ)
  • fluorescence-activated cell sorting FACS
  • OctetTM FormetTM
  • Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with an antigen or a receptor, or ligand binding portion thereof, that specifically binds with a cognate ligand or binding partner.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background, even more specifically, an antibody is said to "specifically bind" an antigen when the equilibrium dissociation constant (K D ) is ⁇ 1 ⁇ M, preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • K D equilibrium dissociation constant
  • ADCC antibody dependent cell-mediated cytotoxicity
  • PBMCs peripheral blood mononuclear cells
  • NK natural killer cells
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-656 .
  • ADCP antibody dependent cell-mediated phagocytosis
  • cytotoxic cells that express Fc gamma Rs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.
  • CDC or "complement dependent cytotoxicity” refer to the lysing of a target cell in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen.
  • a CDC assay such as, but not limited to, an assay described in Gazzano-Santoro et al., 1996, J. Immnol. Methods 202:163 , may be performed.
  • the "CH2 domain" of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain.
  • the "CH3 domain” comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to about amino acid residue 447 of an IgG).
  • effector function refers to the biological activities attributable to or mediated by the Fc region of an antibody.
  • exemplary "effector functions” include, but are not limited to, C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody dependent cell-mediated phagocytosis (ADCP); down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. See, e.g., U.S. Patent No. 6,737,056 .
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays as herein disclosed, for example, as well as those assays known in the art, for evaluating such antibody effector functions.
  • a binding domain e.g., an antibody variable domain
  • Fc fusion or “Fc fusion protein” as used herein is meant a protein wherein one or more polypeptides is operably linked to an Fc region or a derivative thereof.
  • Fc fusion is herein meant to be synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes) as used in the prior art ( Chamow et al., 1996, Trends Biotechnol. 14:52-60 ; Ashkenazi et al., 1997, Curr. Opin. Immunol. 9:195-200 ).
  • An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein or small molecule.
  • the role of the non-Fc part of an Fc fusion, i.e. the fusion partner, is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody.
  • Virtually any protein or small molecule may be linked to an Fc polypeptide to generate an Fc fusion.
  • Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain.
  • Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target.
  • Such targets may be any molecule, preferably an extracellular receptor, that is implicated in disease.
  • IgG immunoglobulin gamma gene
  • this class comprises IgG1, IgG2, IgG3, and IgG4.
  • mice this class comprises IgG1, IgG2a, IgG2b, IgG3.
  • immunoglobulin (Ig) herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies. Immunoglobulins may have a number of structural forms, including but not limited to full length antibodies, antibody fragments, and individual immunoglobulin domains.
  • immunoglobulin (Ig) domain a region of an immunoglobulin that exists as a distinct structural entity as ascertained by one skilled in the art of protein structure. Ig domains typically have a characteristic folding topology.
  • the known Ig domains in the IgG class of antibodies are the variable heavy chain domain (V H ), the heavy chain constant domains - C ⁇ 1, C ⁇ 2, C ⁇ 3 - together comprising the C ⁇ domain which includes the hinge region between C ⁇ 1 and C ⁇ 2, the variable domain of the light chain (V L ), and and the constant domain of the light chain (C L ), which in humans comprises either the kappa (C ⁇ ) or lambda (C ⁇ ) light chain constant domain
  • an "Fc polypeptide” comprises a C ⁇ 2 and a C ⁇ 3 domain and can include at least a portion of the hinge domain, but does not usually include the entire C ⁇ 1 domain.
  • parent polypeptide or “precursor polypeptide” (including Fc parent or precursors) as used herein is meant a polypeptide that is subsequently modified to generate a variant or mutant.
  • Said parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide.
  • Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, to the amino acid sequence of the polypeptide, or to the nucleic acid sequence that encodes it.
  • parent Fc polypeptide as used herein is meant an unmodified Fc polypeptide that is modified to generate a variant
  • parent antibody as used herein is meant an unmodified antibody that is modified to generate a variant antibody.
  • wild-type amino acid refers to a sequence of amino or nucleic acids that occurs naturally within a certain population (e.g., human, mouse, rats, cell, etc.).
  • positions of the Fc molecule can be altered.
  • position as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index as in Kabat. For example, position 297 is a position in the human antibody IgG1. Corresponding positions are determined as outlined above, generally through alignment with other parent sequences.
  • residue as used herein is meant a position in a protein and its associated amino acid identity.
  • Asparagine 297 also referred to as Asn297, also referred to as N297
  • Asn297 is a residue in the human antibody IgG1.
  • target antigen as used herein is meant the molecule that is bound specifically by the variable region of a given antibody.
  • a target antigen may be a protein, carbohydrate, lipid, or other chemical compound.
  • target cell as used herein is meant a cell that expresses a target antigen.
  • variable region as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the V kappa, V.lamda., and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively.
  • variant polypeptide as used herein is meant a polypeptide sequence that differs from that of a parent polypeptide sequence by virtue of at least one amino acid modification.
  • Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it.
  • the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g., from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.
  • the variant polypeptide sequence herein will preferably possess at least about 80 percent homology with a parent polypeptide sequence, and most preferably at least about 90 percent homology, more preferably at least about 95 percent homology, even more preferably, at least about 97% homology, more preferably, at least about 98% homology, and yet more preferably, at least about 99% homology with a parent polypeptide sequence.
  • Fc variant as used herein is meant an Fc sequence that differs from that of a parent Fc sequence by virtue of at least one amino acid modification.
  • An Fc variant may only encompass an Fc region, or may exist in the context of an antibody, Fc fusion, or other polypeptide that is substantially encoded by Fc.
  • Fc variant may refer to the Fc polypeptide itself, compositions comprising the Fc variant polypeptide, the amino acid sequence of the Fc polypeptide, or the nucleic acid sequence that encodes it.
  • the variant proteins of the invention comprise an Fc variant, as described herein, and as such, may comprise an antibody (and the corresponding derivatives) with the Fc variant, or an Fc fusion protein that comprises the Fc variant.
  • the Fc is a variant as compared to a wild-type Fc, or to a "parent" variant.
  • the numbering system used for the light chain constant region amino acid sequence is that set forth in Kabat 1991.
  • substantially pure means an object species is the predominant species present ( i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species (e.g., a glycoprotein, including an antibody or receptor) comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
  • a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • to "treat” means reducing the frequency with which symptoms of a disease (i.e., tumor growth and/or metastasis, or other effect mediated by the numbers and/or activity of immune cells, and the like) are experienced by a patient.
  • the term includes the administration of the compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • “Combination therapy” embraces the administration of an antibody-drug conjugate, and another therapeutic agent as part of a specific treatment regimen optionally including a maintenance phase, intended to provide a beneficial effect from the co-action of these therapeutic agents.
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).
  • “Combination therapy” generally is not intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.
  • Combination therapy embraces administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular, subcutaneous routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents can be administered by the same route or by different routes.
  • a first therapeutic agent e.g., a chemotherapeutic agent
  • a second agent e.g., an ADC
  • a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally.
  • both the therapeutic agents may be administered by intravenous or subcutaneous injection.
  • sequential means, unless otherwise specified, characterized by a regular sequence or order, e.g., if a dosage regimen includes the administration of an ADC and a chemotherapeutic agent, a sequential dosage regimen could include administration of the ADC before, simultaneously, substantially simultaneously, or after administration of the chemotherapeutic agent, but both agents will be administered in a regular sequence or order.
  • sequential dosage regimen means, unless otherwise specified, to keep apart one from the other.
  • simultaneous means, unless otherwise specified, happening or done at the same time, i.e., the compounds of the invention are administered at the same time.
  • substantially simultaneously means that the compounds are administered within minutes of each other (e.g., within 10 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but if the administration is consecutive it is separated in time for only a short period (e.g., the time it would take a medical practitioner to administer two compounds separately).
  • concurrent administration and substantially simultaneous administration are used interchangeably.
  • Sequential administration refers to temporally separated administration of the ADC and the chemotherapeutic agent.
  • Combination therapy also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent, a dendritic cell vaccine or other tumor vaccine) and non-drug therapies (such as, but not limited to, surgery or radiation treatment).
  • the combination therapy further comprises radiation treatment
  • the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the protein or portion thereof, may also be included in the composition.
  • the disclosure is based on the finding that certain residues presumably present on the surface of the CH2 or CH3 domain of the heavy chain of antibodies, or on the constant domain of the light chain, or otherwise accessible, are suitable for the substitution of the naturally-occurring wild type amino acid with, for example, cysteine, and are therefore useful to engineer a site capable of conjugation to various agents.
  • amino acids besides cysteine may be used in the substitution to allow, among other things, for conjugation of various agents.
  • Such other amino acids include lysine (described in Benhar et al., (1994) Bioconjug. Chem. 55:321-326 ), tyrosine (described in Byers and Baldwin, (1988) Immunol. 65:329-335 ), histidine (described in Waibel et al. (1999) Nature Biotechnol. 17:897-901 ), selenocysteine, selenomethionine, and/or non-natural amino acids.
  • cysteine substitutions are described herein, one of ordinary skill in the art may optionally employ one or more of these natural and/or non-natural amino acids instead of cysteine.
  • One of ordinary skill in the art may also use any combination of amino acids in the substitution, such as substituting with cysteine and lysine to produce a variant antibody with cysteines substituted at some positions and lysines, tyrosines, histidines, selenocysteines, selenomethionines, and/or non-natural amino acids at others in any combination thereof.
  • Amino acid modifications can be made by any method known in the art and many such methods are well known and routine for the skilled artisan. For example, but not by way of limitation, amino acid substitutions, deletions and insertions may be accomplished using any well-known PCR-based technique. Amino acid substitutions may be made by site-directed mutagenesis (see, for example, Zoller and Smith, 1982, Nucl. Acids Res. 10:6487-6500 ; Kunkel, 1985, Proc. Natl. Acad. Sci USA 82:488 ).
  • the engineered Fc polypeptide of the disclosure may be used to prepare an antibody, or antigen binding fragment thereof, such that the antibody or fragment thereof thereby comprises an engineered Fc region which can be used to conjugate, at the engineered residue (i.e., the amino acid substituted compared to wild type unmodified Fc), a wide variety of moieties.
  • the engineered kappa light chain constant polypeptide of the disclosure may be used to prepare an antibody, or antigen binding fragment thereof, such that the antibody or fragment thereof thereby comprises an engineered C L region comprising an amino acid mutation, or portion thereof, which can be used to conjugate, at the engineered amino acid residue, a wide variety of moieties.
  • the disclosure provides disclosurean engineered C ⁇ , polypeptide, including, but not limited to, an Fc polypeptide, where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids chosen from positions: 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the antibody heavy chain wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al.
  • Kabat (1991, NIH Publication 91- 3242, National Technical Information Service, Springfield, VA, hereinafter "Kabat") of a parent, native, or wild type antibody, are substituted with another amino acid (including natural and non-natural/synthetic amino acids).
  • the resultant engineered IgG antibodies of the disclosure may display at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more reactive groups for the purpose of conjugation to a drug or compound.
  • one or more of the substitutions is with a cysteine residue
  • the resulting engineered antibodies may display at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more thiol groups for the purpose of conjugation to a drug or compound.
  • the engineered antibody comprises one engineered Fc polypeptide comprising different substituted positions from a second engineered Fc region. That is, because of the dimeric nature of IgG antibodies and because a variety of art-recognized methods for preparing heterodimeric antibodies comprising, inter alia, two or more Fc regions that differ from each other, the present disclosure encompasses an antibody comprising at least one engineered Fc region comprising an amino acid substitution that is not present in the other Fc region, which may or may not also be engineered. Methods for making heterodimeric antibodies comprising Fc regions comprising different mutations are well-known in the art and include, but are not limited to, the methods discussed in U.S. Patent No. 7,183,076 to Arathoon et al.
  • an engineered antibody comprises a first engineered Fc polypeptide comprising at least one substitution at positions selected from 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, and further comprises a second Fc region that is not engineered, e.g., it comprises the amino acid sequence of wild type IgG1.
  • an engineered antibody comprises a first engineered Fc polypeptide comprising at least one substitution at positions selected from 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, and further comprises a second engineered Fc polypeptide that comprises at least one substitution at positions selected from 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334
  • the engineered Fc polypeptide of the disclosure comprises at least one substitution at positions selected from: 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the heavy chain of an antibody, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (supra).
  • the engineered Fc polypeptide of the disclosure comprises at least two substitutions at positions selected from: 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the heavy chain of an antibody, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat et al. (supra).
  • the engineered Fc polypeptide of the disclosure comprises at least two substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least three substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least four substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least five substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least six substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least seven substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least eight substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least nine substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprises at least ten substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 359, 360, 361, 362, 370, 373, 376, 378, 380, 382, 383, 384, 386, 388, 390, 392, 393, 398, 401, 404, 411, 413, 414, 416, 418, 419, 421, 422, 428, 431, 432, 437, 438, 439, 440, 443, and 444, of the heavy chain of an antibody, wherein at least one substitution is selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302,
  • the engineered Fc polypeptide of the disclosure comprise substitutions at each of the positions 254, 359, 361, 380, 383, 384, 392, 398, 404, 422, 442, and 443 of the heavy chain of an antibody wherein the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • the engineered Fc polypeptide of the disclosure comprises at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs:97-100, 102, 104, 107-127, 129-163.
  • the engineered Fc polypeptide of the disclosure comprises at least two amino acid sequences selected from the amino acid sequence of SEQ ID NOs:97-100, 102, 104, 107-127, and 129-163.
  • the engineered Fc polypeptide of the disclosure comprises at least one pair of amino acid sequences selected from: (a) the amino acid sequence of SEQ ID NO:99 and the amino acid sequence of SEQ ID NO:107; (b) the amino acid sequence of SEQ ID NO:103 and the amino acid sequence of SEQ ID NO:107; (c) the amino acid sequence of SEQ ID NO:105 and the amino acid sequence of SEQ ID NO:107; (d) the amino acid sequence of SEQ ID NO:99 and the amino acid sequence of SEQ ID NO:103; (e) the amino acid sequence of SEQ ID NO:103 and the amino acid sequence of SEQ ID NO:105; (f) the amino acid sequence of SEQ ID NO:99 and the amino acid sequence of SEQ ID NO:105; (g) the amino acid sequence of SEQ ID NO:102 and the amino acid sequence of SEQ ID NO:107; (h) the amino acid sequence of SEQ ID NO:104 and the amino acid sequence of SEQ ID NO:107; and (i) the amino acid sequences selected from: (a
  • the engineered C ⁇ polypeptide of the disclosure comprises at least one amino acid sequence selected from the group consisting of the sequence of SEQ ID NOs:90-95.
  • the engineered C ⁇ polypeptide of the disclosure comprises at least one amino acid sequence selected from the group consisting of the sequence of SEQ ID NOs:164-169.
  • an antibody of the disclosure may comprise at least one engineered Fc region and may comprise two engineered Fc regions, where each engineered Fc region may comprise the same or different mutations. More preferably, both engineered Fc regions comprise the same mutations thus providing at least one site-specific conjugation site per each Fc region.
  • an antibody of the disclosure may comprise at least one engineered light chain constant polypeptide (e.g., C ⁇ or C ⁇ ) and may comprise two engineered light chain constant polypeptides, where each engineered light chain constant polypeptide may comprise the same or different mutations. More preferably, both engineered light chain constant polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each light chain constant region.
  • engineered light chain constant polypeptide e.g., C ⁇ or C ⁇
  • both engineered light chain constant polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each light chain constant region.
  • an antibody of the disclosure may comprise at least one engineered Fc polypeptide and may further comprise at least one engineered light chain constant polypeptide thereby providing at least two site-specific conjugation sites - one in the Fc polypeptide and another in the C L polypeptide.
  • an antibody of the disclosure may comprise two engineered Fc polypeptides, where each engineered Fc may comprise the same or different mutations and the antibody further comprises at least one engineered light chain constant region (C ⁇ or C ⁇ ) polypeptide comprising at least one mutation.
  • the antibody comprises two engineered Fc polypeptides comprising at least one mutation, and further comprises two engineered light chain constant (C ⁇ or C ⁇ ) polypeptides each comprising at least one mutation thereby providing at least four site-specific conjugation sites - one per heavy chain and one per light chain. More preferably, both engineered Fc polypeptides comprise the same mutation relative to each other and both light chain constant region (C ⁇ or C ⁇ ) polypeptides comprise the same mutation relative to each other.
  • an antibody of the disclosure may comprise two engineered Fc polypeptides, where each engineered Fc may comprise the same or different mutations and the antibody further comprises at least one engineered light chain constant region (C ⁇ or C ⁇ ) polypeptide comprising at least one mutation.
  • the antibody comprises two engineered Fc polypeptides comprising at least one mutation, and further comprises two engineered light chain constant (two C ⁇ , two C ⁇ or one C ⁇ and one C ⁇ ) polypeptides each comprising at least one mutation, wherein the mutation may be the same or different between the two light chain constant domains) thereby providing at least four site-specific conjugation sites - one per heavy chain and one per light chain.
  • the disclosure encompasses a bispecific antibody comprising two different heavy chains and two different light chains such that the antibody binds, e.g., two different antigens or different epitopes of the same antigen, and wherein the heavy chains comprise at least one engineered Fc and/or one engineered light chain constant domain.
  • the antibody comprises two different heavy chains each comprising the same or different engineered cysteine mutations and one lambda light chain and one kappa light chain wherein each light chain may comprise at least one engineered cysteine mutation.
  • an engineered Fab may comprise at least one mutation in the light chain constant region (C ⁇ or C ⁇ ) to provide at least one site-specific conjugation site thereby providing a Fab comprising at least one site-specific conjugation site.
  • the disclosure encompasses an engineered F(ab') 2 wherein at least one light chain constant region (C ⁇ or C ⁇ ) comprises at least one mutation thereby providing a Fab comprising at least one site-specific conjugation site.
  • the engineered F(ab') 2 of the disclosure comprises at least one mutation in each light chain constant region (C ⁇ or C ⁇ ) thereby providing an engineered F(ab') 2 comprising at least two site-specific conjugation sites.
  • the antibody comprises one engineered Fc polypeptide comprising at least one mutation and two engineered light chain C ⁇ polypeptides each comprising at least one mutation.
  • an antibody of the disclosure may comprise two engineered Fc polypeptides and two engineered (C ⁇ or C ⁇ ) polypeptides where each Fc and each C L comprises at least one mutation and where the Fc region mutation is the same in each Fc polypeptide and the mutation in one C L (C ⁇ or C ⁇ ) polypeptide is different from the mutation in the other C L (C ⁇ or C ⁇ ) polypeptide.
  • each of the mutations in the two Fc regions may be the same, each of the mutations in the C L may the same, or each Fc region and/or each C L (C ⁇ or C ⁇ ) comprises a different mutation, or any permutation thereof.
  • the engineered Fc polypeptide of the disclosure may be used to prepare an Fc fusion protein such that the Fc fusion protein comprises an engineered Fc polypeptide which can be used to conjugate a wide plethora of moieties to the Fc polypeptide.
  • the disclosure encompasses dimeric Fc fusion proteins comprising at least two engineered Fc polypeptides, where each engineered Fc polypeptide may comprise at least one mutation providing a site specific for conjugation.
  • the disclosure provides engineered Fc polypeptide comprising one of the following pairs of substitutions at positions: a) 380 and 443; b) 398 and 443; c) 422 and 443; d) 380 and 398; e) 398 and 442; f) 380 and 422; g) 392 and 443; h) 404 and 443; and i) 392 and 404.
  • the disclosure provides engineered Fc polypeptide comprising at least one of the following pairs of substitutions at positions: a) 380 and 443; b) 398 and 443; c) 422 and 443; d) 380 and 398; e) 398 and 442; f) 380 and 422; g) 392 and 443; h) 404 and 443; and i) 392 and 404.
  • substitutions described above correspond to the positions in SEQ ID NO: 1 (wild type human IgG1 Fc region), and it is intended that the number with reference to the Eu index numbering system of Edelman et al., 1969, as described in Kabat 1991 throughout the disclosure may be used interchangeably with the sequential position numbering of the substitutions in reference with SEQ ID NO: 1 to describe the compositions of the disclosure.
  • the engineered Fc polypeptide of the disclosure comprises a substitution of at least one naturally occurring amino acid chosen from: K246, D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, N315, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, Q386, E388, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, Q419, N421, M428, A431, L432, T437, Q438, K439, L443, and S444 where the numbering is based on the heavy chain of an antibody using the numbering system of the EU index as set forth in Kabat.
  • the engineered Fc polypeptide of the disclosure does not comprise a substitution at a position or positions selected from: 239, 254, 284, 287, 327, 361, 383, 384, 398, 422 and 440 of the heavy chain of an antibody wherein the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • the engineered Fc polypeptide of the disclosure includes an IgG1 having a naturally occurring amino acid substituted (for example, with a cysteine) at a position chosen from: 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the heavy chain of an antibody wherein the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • the engineered Fc polypeptide of the disclosure is derived from an IgG1, IgG2, IgG3 or an IgG4 format. In yet other embodiments, the engineered Fc polypeptide of the disclosure is derived from non-IgG formats such as IgA1, IgA2 IgM, IgD, or IgE. In other embodiments, Fc polypeptide of the disclosure comprise engineering of surface residues of the CH2 and/or CH3 region of an IgG1 molecule or equivalents thereof by substitution of a naturally-occurring residue for cysteine and/or other amino acids.
  • the disclosure encompasses an engineered antibody light chain constant region (C L ) wherein the light chain is a kappa light chain or a lambda light chain, or a portion thereof, where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids chosen from positions 111, 149, 183, 188, 207, and 210 of the antibody light chain, wherein the numbering system of the light chain constant region is that of the Kabat numbering system as set forth in Kabat et al.
  • Kabat (1991, NIH Publication 91- 3242, National Technical Information Service, Springfield, VA, hereinafter "Kabat"), of a parent, native, or wild type antibody, are substituted with another amino acid (including natural and non-natural/synthetic amino acids).
  • the light chain constant region is a lambda constant region (CA). In another embodiment, the light chain constant region is a kappa constant region (C ⁇ ).
  • the resultant engineered IgG antibodies of the disclosure may display at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more reactive groups for the purpose of conjugation to a drug or compound.
  • one or more of the substitutions is with a cysteine residue
  • the resulting engineered antibodies may display at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more thiol groups for the purpose of conjugation to a drug or compound.
  • the disclosure provides an engineered antibody comprising one engineered polypeptide comprising a kappa light chain constant domain (C ⁇ ) comprising the same substituted position from a second engineered kappa light chain constant domain. That is, because of the dimeric bivalent nature of IgG antibodies, the antibody may comprise two C ⁇ polypeptides that are the same or differ from each other, and the present disclosure encompasses an antibody comprising at least one engineered C ⁇ polypeptide comprising an amino acid substitution that is present in the other C ⁇ polypeptide.
  • C ⁇ kappa light chain constant domain
  • the engineered antibody comprises one engineered polypeptide comprising a kappa light chain constant domain (C ⁇ ) comprising different substituted positions from a second engineered kappa light chain constant domain.
  • C ⁇ kappa light chain constant domain
  • an engineered antibody of the disclosure comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 111, 149, 183, 188, 207, and 210, and further comprises a second C ⁇ polypeptide that is not engineered, e.g., it comprises the amino acid sequence of wild type C ⁇ where an exemplary human wild type C ⁇ polypeptide amino acid sequence is shown in Figure 18A and is provided in SEQ ID NO:89.
  • an engineered Fab of the disclosure comprises an engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 111, 183, and 210.
  • an engineered antibody comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 111, 183, and 210, and further comprises a second engineered C ⁇ polypeptide that comprises at least one substitution at positions selected from 111, 183, and 210, wherein the substitution present in the first engineered C ⁇ polypeptide is not a substitution present in the second engineered C ⁇ polypeptide.
  • an engineered F(ab') 2 of the disclosure comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 111, 149, 183, 188, 207, and 210, and further comprises a second engineered C ⁇ polypeptide that comprises at least one substitution at positions selected from 111, 183, and 210, wherein the substitution present in the first engineered C ⁇ polypeptide is not a substitution present in the second engineered C ⁇ polypeptide.
  • an engineered F(ab') 2 of the disclosure comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 111, 149, 183, 188, 207, and 210, and further comprises a second engineered C ⁇ polypeptide that comprises at least one substitution at positions selected from 111, 183, and 210, wherein at least one substitution present in the first engineered C ⁇ polypeptide is the same substitution present in the second engineered C ⁇ polypeptide.
  • the engineered C ⁇ polypeptide of the disclosure comprises at least one substitution at positions selected from: 111, 183, and 210 of the light chain of an antibody, wherein the numbering system of the constant region is that of the Kabat index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least two substitutions at positions selected from: 111, 183, and 210 of the light chain of an antibody, wherein the numbering system of the constant region is that of the Kabat index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises all three substitutions selected from the positions 111, 183, and 210, of the light chain constant region of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • substitutions described above correspond to the positions in SEQ ID NO:89 (wild type human kappa light chain constant region), and it is intended that the number with reference to Kabat throughout the disclosure may be used interchangeably with the sequential position numbering of the substitutions in reference with SEQ ID NO:89 to describe the compositions of the disclosure.
  • the engineered C ⁇ polypeptide of the disclosure further comprises a substitution of at least one naturally occurring amino acid chosen from: A111, K183, and N210 where the numbering is based on the light chain of an antibody using the numbering system of the Kabat numbering index as set forth in Kabat.
  • the engineered light chain constant domain is a lambda light chain constant domain (C ⁇ ).
  • the engineered antibody comprises one engineered polypeptide comprising a lambda light chain constant domain (C ⁇ ) comprising the same or different substituted positions from a second engineered lambda light chain constant domain.
  • C ⁇ lambda light chain constant domain
  • the engineered antibody comprises at least one engineered polypeptide comprising a lambda light chain constant domain (C ⁇ ), wherein the antibody comprises two C ⁇ domains each comprising the same mutation or mutations.
  • C ⁇ lambda light chain constant domain
  • an engineered antibody of the disclosure comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from K110C, L125C, K149C, V155C, G158C, T161C, Q185C, S188C, H189C, S191C, T197C, V205C, E206C, K207C, T208 and A210 and further comprises a second C ⁇ polypeptide that is not engineered, e.g., it comprises the amino acid sequence of wild type C ⁇ where an exemplary human wild type C ⁇ polypeptide amino acid sequence is shown in Figure 20A and is provided in SEQ ID NO:170.
  • an engineered Fab of the disclosure comprises an engineered C ⁇ polypeptide comprising at least one substitution at positions selected from K110, L125, K149, V155, G158, T161, Q185, S188, H189, S191, T197, V205, E206, K207C, T208 and A210.
  • an engineered antibody comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, K207C, T208 and A210 and further comprises a second engineered C ⁇ polypeptide that comprises at least one substitution at positions selected from 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, K207C, T208 and A210, wherein the substitution present in the first engineered C ⁇ polypeptide is not a substitution present in the second engineered C ⁇ polypeptide.
  • an engineered F(ab') 2 of the disclosure comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210 and further comprises a second engineered C ⁇ polypeptide that comprises at least one substitution at positions selected from 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210 wherein the substitution present in the first engineered C ⁇ polypeptide is not a substitution present in the second engineered C ⁇ polypeptide.
  • an engineered F(ab') 2 of the disclosure comprises a first engineered C ⁇ polypeptide comprising at least one substitution at positions selected from 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, and further comprises a second engineered C ⁇ polypeptide that comprises at least one substitution at positions selected from 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, wherein at least one substitution present in the first engineered C ⁇ polypeptide is the same substitution present in the second engineered C ⁇ polypeptide.
  • the engineered C ⁇ polypeptide of the disclosure comprises at least one substitution at positions selected from: 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain of an antibody, wherein the numbering system of the constant region is that of the Kabat index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least two substitutions at positions selected from: 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 20C, 208 andA210, of the light chain of an antibody, wherein the numbering system of the constant region is that of the Kabat index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least two substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain constant region of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least three substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain constant region of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least four substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207C, 208 and 210, of the light chain constant region of an antibody, wherein at least one substitution is selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210 of the light chain constant domain of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least five substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain constant region of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least six substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least seven substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least eight substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least nine substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207C, 208 and 210, of the light chain of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises at least ten substitutions selected from the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain of an antibody, and wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat et al. (supra).
  • the engineered C ⁇ polypeptide of the disclosure comprises substitutions at each of the positions 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210, of the light chain of an antibody wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat.
  • substitutions described above correspond to the positions in SEQ ID NO:170 (wild type human lambda light chain constant region), and it is intended that the number with reference to Kabat throughout the disclosure may be used interchangeably with the sequential position numbering of the substitutions in reference with SEQ ID NO:170 to describe the compositions of the disclosure.
  • the engineered C ⁇ polypeptide of the disclosure comprises a substitution of at least one naturally occurring amino acid chosen from: 110, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208 and 210 where the numbering is based on the light chain of an antibody using the numbering system of the Kabat numbering index as set forth in Kabat.
  • an antibody of the disclosure may comprise at least one engineered constant region (e.g., a heavy chain constant region, an IgG C ⁇ region, a C ⁇ region, and/or a C ⁇ region) and may comprise two engineered C L regions, where each engineered C L region may comprise the same or different mutations. More preferably, both engineered C L (C ⁇ or C ⁇ ) regions comprise the same mutations thus providing at least one site-specific conjugation site per each C L (C ⁇ or C ⁇ ) region.
  • an engineered constant region e.g., a heavy chain constant region, an IgG C ⁇ region, a C ⁇ region, and/or a C ⁇ region
  • engineered C L regions comprise the same mutations thus providing at least one site-specific conjugation site per each C L (C ⁇ or C ⁇ ) region.
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide and may comprise two engineered Fc polypeptides, where each engineered Fc polypeptide may comprise the same or different mutations. More preferably, both engineered Fc polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each Fc polypeptide.
  • the antibody may further comprise at least one engineered C ⁇ polypeptide and may comprise two engineered C ⁇ polypeptides wherein each engineered C ⁇ polypeptide may comprise the same or different mutations.
  • both engineered Fc polypeptides comprise the same mutations and both engineered C ⁇ polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each Fc polypeptide and at least one site-specific conjugation site per C ⁇ polypeptide thereby providing an antibody comprising at least four potential conjugation sites.
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide and may comprise two engineered Fc polypeptides, where each engineered Fc polypeptide may comprise the same or different mutations. More preferably, both engineered Fc polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each Fc polypeptide.
  • the antibody may further comprise at least one engineered C ⁇ polypeptide and may comprise two engineered C ⁇ polypeptides wherein each engineered C ⁇ polypeptide may comprise the same or different mutations.
  • both engineered Fc polypeptides comprise the same mutations and both engineered C ⁇ polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each Fc polypeptide and at least one site-specific conjugation site per C ⁇ polypeptide thereby providing an antibody comprising at least four potential conjugation sites.
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide and may comprise two engineered Fc polypeptides, where each engineered Fc polypeptide may comprise the same or different mutations. More preferably, both engineered Fc polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each Fc polypeptide.
  • the antibody may further comprise at least one engineered C ⁇ polypeptide and may comprise two engineered C ⁇ polypeptides wherein each engineered C ⁇ polypeptide may comprise the same or different mutations.
  • both engineered Fc polypeptides comprise the same mutations and both engineered C ⁇ polypeptides comprise the same mutations thus providing at least one site-specific conjugation site per each Fc polypeptide and at least one site-specific conjugation site per C ⁇ polypeptide thereby providing an antibody comprising at least four potential conjugation sites.
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide comprising at least one substitution selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of the EU index as set forth in Kabat, and comprises at least one engineered C ⁇ polypeptide comprising at least one substitution selected from the positions 111, 149, 183, 188, 207, and 210, of the
  • the engineered antibody comprises at least one engineered heavy chain constant domain (C ⁇ ) comprising at least one amino acid substitution selected from a substitution at position 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, according to the Eu numbering of Kabat, and/or where the antibody comprises at least one engineered C ⁇ domain comprising at least one amino acid substitution selected from the group consisting of K110C, L125C, K149C, V155C,
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide comprising at least one substitution selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of the EU index as set forth in Kabat, and comprises at least one engineered C ⁇ polypeptide comprising at least one substitution selected from the positions 110, 111, 125, 149, 155, 158, 161, 185
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide comprising at least one substitution selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of the EU index as set forth in Kabat, and comprises at least one: (a) engineered C ⁇ polypeptide comprising at least one substitution selected from the positions 111, 183, and 210, of the light chain of an antibody
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide comprising at least one substitution selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of the EU index as set forth in Kabat, and comprises at least one: (a) engineered C ⁇ polypeptide comprising at least one substitution selected from the positions 111, 149, 183, 188, 207, and
  • the engineered antibody of the disclosure comprises at least one engineered engineered C ⁇ polypeptide comprising at least one substitution selected from the positions 111, 149, 183, 188, 207, and 210, of the light chain of an antibody, and comprises at least one: (a) Fc polypeptide comprising at least one substitution selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of
  • the engineered antibody of the disclosure comprises at least one engineered engineered C ⁇ polypeptide comprising at least one substitution selected from the positions 111, 149, 183, 188, 207, and 210, of the light chain of an antibody wherein the numbering is according to Kabat, and an Fc polypeptide comprising at least one substitution selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using
  • the engineered antibody of the disclosure comprises at least one engineered engineered C ⁇ polypeptide comprising at least one substitution selected from the positions 110, 111, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206 and 207, wherein the numbering system of the light chain constant polypeptide is that of the Kabat numbering index as set forth in Kabat et al., and comprises at least one: (a) Fc polypeptide comprising at least one substitution selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411,
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide comprising at least two substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of the EU index as set forth in Kabat, and comprises at least one engineered C ⁇ polypeptide comprising at least two substitutions selected from the positions 111, 149, 183, 188, 207, and 210,
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide comprising at least three substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of the EU index as set forth in Kabat, and comprises at least one engineered C ⁇ polypeptide comprising at least three substitutions selected from the positions 111, 149, 183, 188, 207, and 210,
  • the engineered antibody of the disclosure comprises at least one engineered Fc polypeptide comprising at least five substitutions selected from the positions 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, of the heavy chain using the numbering system of the EU index as set forth in Kabat, and comprises at least one engineered C ⁇ polypeptide comprising at least five substitutions selected from the positions 111, 149, 183, 188, 207, and 210,
  • the antibody comprises at least two engineered Fc polypeptides and two engineered light chain constant polypeptides (C ⁇ -C ⁇ , C ⁇ -C ⁇ or C ⁇ -C ⁇ ) wherein each of the mutations in the two Fc polypeptides may be the same, each of the mutations in the C ⁇ or C ⁇ may the same, or each Fc polypeptide and/or each C ⁇ or C ⁇ comprises a different mutation, or no mutation, and any combination of the foregoing.
  • C ⁇ -C ⁇ , C ⁇ -C ⁇ or C ⁇ -C ⁇ two engineered light chain constant polypeptides
  • One of ordinary skill in the art can readily select a suitable amino acid to use in the substitution. It may be desirable to select a residue that is similar to the non-naturally occurring residue (e.g., a conservative substitution) in order to minimize changes to the protein structure. For example, for cysteine substitutions, it can be desirable, but not necessary, to substitute cysteine for a naturally occurring alanine or serine.
  • substitutions in IgG2, IgG3, and IgG4 one of ordinary skill in the art can use sequence alignment of the Ig type of interest with IgG1 to determine the relative residues of the desired isoform corresponding with the above-described positions of positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the heavy chain of an antibody wherein the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • HC Fc human wild type heavy chain constant domains
  • FIG 19A An exemplary alignment, showing the corresponding positions for each amino acid of the Fc domains of human IgG1, IgG2, IgG3 and IgG4, is provided in Figure 19A .
  • the Fc region for human IgG1 begins at amino acid residue 236 glycine ( 236 G) using the Eu index as described in Kabat.
  • the sequences shown in Figure 15 show the CH1 region and the complete hinge region of human IgG1 where the human Fc region more preferably begins at 236 G.
  • the disclosure encompasses expression of an isolated Fc polypeptide comprising engineered residues.
  • isolated Fc polypeptides may be useful as scaffolds for display purposes or as dimerization domains alone or when combined with another agent.
  • the disclosure comprises a fusion protein comprising an engineered Fc polypeptide and a binding domain comprising a binding site of a receptor, cytokine, ligand, and the like, such that the binding domain provides binding specificity for the engineered Fc polypeptide such that any moiety conjugated to the engineered Fc polypeptide is targeted to the cognate binding molecule that specifically binds with the binding domain.
  • An exemplary Fc fusion protein encompassed by the disclosure comprises a tumor necrosis factor receptor 2 (TNFR2), or a TNF ⁇ -binding portion thereof, fused with an engineered Fc polypeptide of the disclosure, similar, but not identical to, etanercept (ENBRELTM), comprising TNFR2 fused with a wild type IgG1 Fc polypeptide.
  • TNFR2 tumor necrosis factor receptor 2
  • ENBRELTM etanercept
  • the disclosure provides fusion proteins comprising an engineered Fc polypeptide that comprises at least one or more substitutions at positions selected from: 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat, fused to another protein.
  • the K D for the antibody with respect to the target will be 2-fold, preferably 5-fold, more preferably 10-fold less than the K D with respect to another, non-target molecule such as, but not limited to, unrelated material or accompanying material in the environment. More preferably, the K D will be 50-fold less, such as 100-fold less, or 200-fold less; even more preferably 500-fold less, such as 1,000-fold less, or 10,000-fold less than the K D with respect the non-target molecule.
  • this dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al., 1984, Byte 9: 340-362 .
  • the K D may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong and Lohman, 1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432 .
  • Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis.
  • the binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a BiacoreTM system.
  • SPR Surface Plasmon Resonance
  • a competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another ligand of that target, such as another antibody.
  • the concentration at which 50 percent binding inhibition occurs is known as the K i .
  • the K i is equivalent to K D .
  • the K i value will never be less than the K D , so measurement of K i can conveniently be substituted to provide an upper limit for K D .
  • An antibody of the invention may have a K D for its target of 1 x 10 -7 M or less, 1 x 10 -8 M or less, or 1 x 10 -9 M or less, or 1 x 10 -10 M or less, 1 x 10 -11 M or less, or 1 x 10 -12 M or less.
  • An antibody that specifically binds its target may bind its target with a high affinity, that is, exhibiting a low K D as discussed above, and may bind to other, non- target molecules with a lower affinity.
  • the antibody may bind to non- target molecules with a K D of 1 x 10 -6 M or more, more preferably 1 x 10 -5 M or more, more preferably 1 x 10 -4 M or more, more preferably 1 x 10 -3 M or more, even more preferably 1 x 10 -2 M or more.
  • An antibody of the invention is preferably capable of binding to its target with an affinity that is at least two-fold, 10-fold, 50-fold, 100- fold 200-fold, 500-fold, 1, 000-fold or 10,000-fold or greater than its affinity for binding to another non-target molecule.
  • an antibody comprising an engineered Fc polypeptide of the disclosure may have an affinity rate constant or K a (k on /k off ) of at least 10 2 M -1 , at least 5 x 10 2 M -1 , at least 10 3 M -1 , at least 5 x 10 3 M -1 , at least 10 4 M -1 , at least 5 x 10 4 M -1 , at least 10 5 M -1 , at least 5 x 10 5 M -1 , at least 10 6 M -1 , at least 5 x 10 6 M -1 , at least 10 7 M -1 , at least 5 x 10 7 M -1 , at least 10 8 M -1 , at least 5 x 10 8 M -1 , at least 10 9 M -1 , at least 5 x 10 9 M -1 , at least 10 10 M -1 , at least 5 x 10 10 M -1 , at least 5 x 10 10 M -1 , at least 10 11 M -1 , at least 5 x 10 11 M
  • an antibody comprising engineered Fc polypeptides of the disclosure may have a dissociation rate constant or K d (k off /k on ) of less than 5 x 10 -2 M, less than 10 -2 M, less than 5 x 10 -3 M, less than 10 -3 M, less than 5 x 10 -4 M, less than 10 -4 M, less than 5 x 10 -5 M, less than 10 -5 M, less than 5 x 10 -6 M, less than 10 -6 M, less than 5 x 10 -7 M, less than 10 -7 M, less than 5 x 10 -8 M, less than 10 -8 M, less than 5 x 10 -9 M, less than 10 -9 M, less than 5 x 10 -10 M, less than 10 -10 M, less than 5 x 10 -11 M, less than 10 -11 M, less than 5 x 10 -12 M, less than 10 -12 M, less than 5 x 10 -13 M, less than 10 -13 M, less than 5 x 10 -14 M
  • An antibody comprising an engineered Fc polypeptide used in accordance with a method described herein may have a dissociation constant (K d ) of less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM as assessed using a method described herein or known to one of skill in the art (e.g., a BIAcore assayTM, ELISA) (Biacore International AB, Uppsala, Sweden).
  • K d dissociation constant
  • Antibodies comprising engineered Fc polypeptides of the disclosure retain the antigen binding capability of their native counterparts.
  • the antibodies comprising an engineered Fc polypeptide of the disclosure exhibit essentially the same affinity as compared to an antibody prior to engineering.
  • antibodies comprising an engineered Fc polypeptide of the disclosure exhibit a reduced affinity as compared to an antibody prior to engineering.
  • antibodies comprising an engineered Fc polypeptide of the disclosure exhibit an enhanced affinity as compared to an antibody prior to engineering.
  • an antibody comprising an engineered Fc polypeptide of the disclosure may have a dissociation constant (K d ) about equal to the K d of the antibody prior to engineering.
  • an antibody comprising an engineered Fc polypeptide of the disclosure may have a dissociation constant (K d ) about 1-fold, more preferably about 2-fold, even more preferably, about 3-fold, more preferably, about 4-fold, yet more preferably, about 5-fold, even more preferably, about 10-fold, more preferably, about 20-fold, even more preferably, about 50-fold, more preferably, about 100-fold, even more preferably, about 150-fold, more preferably, about 200-fold, yet more preferably, about 250-fold, even more preferably, about 300-fold, more preferably, about 400-fold, even more preferably, about 500-fold, more preferably, about 600-fold, even more preferably, about 700-fold, more preferably, about 800-fold, even more preferably 900-fold, and yet more preferably, about 1000-fold greater for its cognate antigen compared with the K d of the antibody prior to engineering.
  • K d dissociation constant
  • an antibody comprising an engineered Fc polypeptide of the disclosure may have a K d about 1-fold, more preferably about 2-fold, even more preferably, about 3-fold, more preferably, about 4-fold, yet more preferably, about 5-fold, even more preferably, about 10-fold, more preferably, about 20-fold, even more preferably, about 50-fold, more preferably, about 100-fold, even more preferably, about 150-fold, more preferably, about 200-fold, yet more preferably, about 250-fold, even more preferably, about 300-fold, more preferably, about 400-fold, even more preferably, about 500-fold, more preferably, about 600-fold, even more preferably, about 700-fold, more preferably, about 800-fold, even more preferably 900-fold, and yet more preferably, about 1000-fold lower for its cognate antigen compared with the K d of the antibody prior to engineering.
  • engineered antibody, Fab, and F(ab') 2 of the disclosure comprises an antibody, Fab, and F(ab') 2 that comprises an epitope binding domain (for example, but not limited to, an antibody variable region having all 6 CDRs, or an equivalent region that is at least 90 percent identical to an antibody variable region) chosen from: abagovomab, abatacept (ORENCIA®), abciximab (REOPRO®, c7E3 Fab), adalimumab (HUMIRA®), adecatumumab, alemtuzumab (CAMPATH®, MabCampath or Campath-1H), altumomab, afelimomab, anatumomab mafenatox, anetumumab, anrukizumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab,
  • an engineered antibody, Fab, and F(ab') 2 of the disclosure comprise a heavy and light chain variable domain having six CDRs, and/or compete for binding with an antibody selected from the preceding list.
  • an antibody, Fab, and F(ab') 2 comprising an engineered Fc polypeptide and/or an engineered C ⁇ or C ⁇ polypeptide of the disclosure bind the same epitope as the antibodies in the preceeding list.
  • an antibody, Fab, and F(ab') 2 comprising an engineered Fc polypeptide and/or an engineered C ⁇ or C ⁇ polypeptide of the disclosure comprises a heavy and light chain variable domain having six total CDRs, and binds to the same antigen as the antibodies in the proceeding list.
  • an antibody, Fab, and F(ab') 2 comprising an engineered Fc polypeptide and/or an engineered C ⁇ or C ⁇ polypeptide of the disclosure comprises a heavy and light chain variable domain having six (6) total CDRs, and specifically binds to an antigen selected from: PDGFRalpha, PDGFRbeta, PDGF, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, VEGFR1, VEGFR2, VEGFR3, FGF, FGF2, HGF, KDR, flt-1, FLK-1, Ang-2, Ang-1, PLGF, CEA, CXCL13, Baff, IL-21, CCL21, TNF-alpha, CXCL12, SDF-I, bFGF, MAC-I, IL23pl9, FPR, IGFBP4, CXCR3, TLR4, CXCR2, EphA2,
  • an antibody, or antigen-binding portion thereof e.g., Fab, and F(ab') 2 fragment, comprising an engineered Fc polypeptide and/or an engineered C ⁇ or C ⁇ polypeptide of the disclosure specifically binds to a member (receptor or ligand) of the TNF superfamily.
  • TNF-alpha Tumor Necrosis Factor-alpha
  • TNF-beta Tumor Necrosis Factor- beta
  • LT-alpha Lymphotoxin-alpha
  • CD30 ligand CD27 ligand, CD40 ligand, 4-1 BB ligand
  • Apo-1 ligand also referred to as Fas ligand or CD95 ligand
  • Apo-2 ligand also referred to as TRAIL
  • Apo-3 ligand also referred to as TWEAK
  • osteoprotegerin OPG
  • APRIL RANK ligand
  • TRANCE also referred to as TRANCE
  • TALL-I also referred to as BlyS, BAFF or THANK
  • DR4 DR5 also known as Apo-2, TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2, or KILLER
  • DR6 DcRI
  • DcR2 DcR3 also known as TR6 or M
  • the antibody, or antigen-binding portion thereof, e.g., Fab, and F(ab') 2 frament, comprising an engineered Fc polypeptide and/or an engineered C ⁇ or C ⁇ polypeptide of the disclosure is capable of binding one or more targets chosen from 5T4, ABL, ABCB5, ABCFI, ACVRI, ACVRIB, ACVR2, ACVR2B, ACVRLI, AD0RA2A, Aggrecan, AGR2, AICDA, AIFI, AIGI, AKAPI, AKAP2, AMH, AMHR2, angiogenin (ANG), ANGPTI, ANGPT2, ANGPTL3, ANGPTL4, Annexin A2, ANPEP, APC, APOCI, AR, aromatase, ATX, AXI, AZGPI (zinc-a-glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAGI, BAII, BCR, BCL2, BCL6, BDNF, BLNK
  • the engineered Fc polypeptide of the disclosure may be fused/covalently linked with a portion of any of the proteins in the preceding list or the Fc polypeptide may be fused/covalently linked with any receptor or ligand that specifically binds a protein in the preceding list.
  • the disclosure encompasses an Fc polypeptide fusion protein comprising an engineered Fc polypeptide fused with a protein listed above, or a portion of the protein that binds its cognate ligand or receptor.
  • abatacept and betanercept are Fc fusion proteins comprising a portion of CTLA4 and TNFR2, respectively.
  • the present disclosure therefore encompasses a fusion protein comprising a CTLA4-Fc fusion protein (abatacept; ORENCIATM) where the Fc is an engineered Fc polypeptide of the disclosure and CTLR4 is an extracellular domain of CTLA4 which is capable of binding its cognate antigens, e.g., CD80 (B7-1) and CD86 (B7-2).
  • CTLA4-Fc fusion protein abatacept; ORENCIATM
  • CTLR4 is an extracellular domain of CTLA4 which is capable of binding its cognate antigens, e.g., CD80 (B7-1) and CD86 (B7-2).
  • the disclosure encompasses a fusion protein comprising a TNFR2-Fc fusion protein that binds TNFalpha (etanercept; ENBRELTM) wherein the Fc is an engineered Fc polypeptide of the disclosure; an Fc fusion protein comprising the extracellular domain (ECD) of LFA3 (alefacept; AMEVIVETM) which binds CD2 wherein the Fc is an engineered Fc polypeptide of the disclosure; and an Fc fusion protein comprising a thrombopoietin receptor-binding peptide which binds thrombopoietin receptor (romiplostim) wherein the Fc is an engineered Fc polypeptide of the disclosure.
  • the disclosure is in no way limited to these particular embodiments, but rather, encompasses a wide variety of Fc fusion proteins comprising any protein of interest fused with an engineered Fc polypeptide of the disclosure.
  • the disclosure encompasses a fusion protein comprising an engineered Fc polypeptide fused with any of the following proteins, or a binding portion thereof: PDGFRalpha, PDGFRbeta, PDGF, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, VEGFR-I, VEGFR-2, VEGFR-3, FGF, FGF2, HGF, KDR, flt-1, FLK-1, Ang-2, Ang-1, PLGF, CEA, CXCL13, Baff, IL-21, CCL21, TNF-alpha, CXCL12, SDF-I, bFGF, MAC-I, IL23pl9, FPR, IGFBP4, CXCR3, TLR4, CXCR2, EphA2, EphA4, EphrinB2, EGFR(ErbBI), HER2(ErbB2 or pl85neu), HER3
  • the Fc fusion comprising an engineered Fc polypeptide of the disclosure is capable of binding one or more proteins chosen from 5T4, ABL, ABCB5, ABCFI, ACVRI, ACVRIB, ACVR2, ACVR2B, ACVRLI, AD0RA2A, Aggrecan, AGR2, AICDA, AIFI, AIGI, AKAPI, AKAP2, AMH, AMHR2, angiogenin (ANG), ANGPTI, ANGPT2, ANGPTL3, ANGPTL4, Annexin A2, ANPEP, APC, APOCI, AR, aromatase, ATX, AXI, AZGPI (zinc-a-glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAGI, BAII, BCR, BCL2, BCL6, BDNF, BLNK, BLRI (MDR15), BlyS, BMPI, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPRIA
  • the disclosure also provides an engineered Fc polypeptide that may be further modified. It is known that variants of the Fc region, e.g., amino acid substitutions, insertions, and/or additions and/or deletions, enhance or diminish effector function. See, e.g., Presta et al, 2002, Biochem. Soc. Trans. 30:487-490 ; Strohl, 2009, Curr. Opin. Biotechnol. 20(6):685-691 ; U.S. patents 5,624,821 , 5,648,260 , 5,885,573 , 6,737,056 , 7,317,091 ; PCT publication Nos.
  • the engineered Fc polypeptide exhibits a similar level of inducing effector function as compared to the native wild-type Fc polypeptide. In another embodiment, the engineered Fc polypeptide exhibits a higher induction of effector function as compared to the native Fc. In another embodiment, the engineered Fc polypeptide exhibits lower induction of effector function as compared to the native Fc. In another embodiment, the engineered Fc polypeptide exhibits higher induction of ADCC as compared to the native Fc. In another embodiment, the engineered Fc polypeptide exhibits lower induction of ADCC as compared to the native Fc. In another embodiment, the engineered Fc polypeptide exhibits higher induction of CDC as compared to the native Fc. In another embodiment, the engineered Fc polypeptide exhibits lower induction of CDC as compared to the native Fc. Specific embodiments of engineered Fc polypeptides further modified to affect effector function are detailed infra.
  • the present disclosure encompasses engineered Fc proteins which further comprise altered binding properties for an Fc ligand (e.g., an Fc receptor, Clq, and the like) relative to a reference molecule (e.g., a protein having the same amino acid sequence except having a native wild type Fc polypeptide).
  • Fc ligand e.g., an Fc receptor, Clq, and the like
  • binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (k off and k on , respectively), binding affinity and/or avidity.
  • K D equilibrium dissociation constant
  • k off and k on respectively
  • binding affinity and/or avidity e.g., avidity
  • a binding molecule e.g., a Fc variant protein such as an antibody
  • the value of the k on or k off may be more relevant than the value of the K D .
  • One skilled in the art can determine which kinetic parameter is most important for
  • the affinities and binding properties of an Fc polypeptide for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-Fc ⁇ R interactions, i.e., specific binding of an Fc polypeptide to an Fc ⁇ R including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis, OCTET®, FortéBio, Menlo Park, CA), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • in vitro assay methods biochemical or immunological based assays
  • ELISA enzyme-linked immunoabsorbent assay
  • RIA radioimmunoassay
  • kinetics
  • These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • the engineered Fc polypeptide comprises an additional mutation and exhibits enhanced binding to one or more Fc ligands relative to a comparable molecule engineered Fc without the additional mutation compared with wild type unmodified Fc.
  • the engineered Fc variant protein has an affinity for an Fc ligand that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold greater than a comparable engineered Fc without the additional mutation.
  • the engineered Fc variant protein has enhanced binding to an Fc receptor.
  • the Fc variant protein has enhanced binding to the Fc receptor Fc ⁇ RIIIA.
  • the Fc variant protein has enhanced binding to the Fc receptor Fc ⁇ RIIB.
  • the Fc variant protein has enhanced binding to the Fc receptor FcRn.
  • the Fc variant protein has enhanced binding to Clq relative to a comparable Fc molecule lacking the mutations (e.g., wild type parental Fc).
  • any particular engineered Fc variant protein to mediate lysis of the target cell by ADCC can be assayed.
  • an engineered Fc variant protein of interest is added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
  • label e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985, J. Immunol.
  • ADCC activity of the engineered Fc variant protein of interest may also be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-656 .
  • an engineered Fc variant protein has enhanced ADCC activity relative to a comparable molecule (e.g., a wild type natural Fc without any mutations and/or an engineered Fc without any additional modifications).
  • a comparable molecule e.g., a wild type natural Fc without any mutations and/or an engineered Fc without any additional modifications.
  • an Fc variant protein has ADCC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold greater than that of a comparable molecule.
  • an Fc variant protein has enhanced binding to the Fc receptor Fc ⁇ RIIIA and has enhanced ADCC activity relative to a comparable molecule.
  • the Fc variant protein has both enhanced ADCC activity and enhanced serum half life relative to a comparable molecule.
  • an engineered Fc variant protein has reduced ADCC activity relative to a comparable molecule (e.g., a wild type natural Fc without any mutations and/or an engineered Fc without any additional modifications).
  • a comparable molecule e.g., a wild type natural Fc without any mutations and/or an engineered Fc without any additional modifications.
  • an engineered Fc variant protein has ADCC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold lower than that of a comparable molecule.
  • an engineered Fc variant protein has reduced binding to the Fc receptor Fc ⁇ RIIIA and has reduced ADCC activity relative to a comparable molecule.
  • the engineered Fc variant protein has both reduced ADCC activity and enhanced serum half life relative to a comparable molecule.
  • an engineered Fc variant protein has enhanced CDC activity relative to a comparable molecule (e.g., a wild type natural Fc without any mutations and/or an engineered Fc without any additional modifications).
  • a comparable molecule e.g., a wild type natural Fc without any mutations and/or an engineered Fc without any additional modifications.
  • an Fc variant protein has CDC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold or at least 50 fold or at least 100 fold greater than that of a comparable molecule.
  • the engineered Fc variant protein has both enhanced CDC activity and enhanced serum half life relative to a comparable molecule.
  • the engineered Fc variant protein has reduced binding to one or more Fc ligand relative to a comparable molecule.
  • the engineered Fc variant protein has an affinity for an Fc ligand that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold lower than that of a comparable molecule.
  • the engineered Fc variant protein has reduced binding to an Fc receptor.
  • the engineered Fc variant protein has reduced binding to the Fc receptor Fc ⁇ RIIIA.
  • an engineered Fc variant described herein has an affinity for the Fc receptor Fc ⁇ RIIIA that is at least about 5 fold lower than that of a comparable molecule, wherein said engineered Fc variant has an affinity for the Fc receptor Fc ⁇ RIIB that is within about 2 fold of that of a comparable molecule.
  • the engineered Fc variant protein has reduced binding to the Fc receptor FcRn.
  • the engineered Fc variant protein has reduced binding to Clq relative to a comparable molecule.
  • glycosylation of an Fc polypeptide can be modified to increase or decrease effector function (see for examples, Uma ⁇ a et al., 1999, Nat. Biotechnol. 17:176-180 ; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294 ; Shields et al., 2002, J. Biol. Chem. 277:26733-26740 ; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473 ; U.S. Pat. No. 6,602,684 ; U.S. Patent Application Publication No. 2003/0157108 ; U.S.
  • Patent Application Publication No. 2003/0003097 International Patent Publication Nos. WO 00/61739A1 ; WO 01/292246A1 ; WO 02/311140A1 ; and WO 02/30954A1 ; PotillegentTM technology (Biowa, Inc. Princeton, N.J.); GlycoMAbTM glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland).
  • the engineered Fc polypeptides of antibodies and fusion proteins of the disclosure may comprise altered glycosylation of amino acid residues.
  • the altered glycosylation of the amino acid residues results in decreased effector function.
  • the altered glycosylation of the amino acid residues results in increased effector function.
  • the engineered Fc polypeptide has reduced fucosylation.
  • the engineered Fc polypeptide is afucosylated (see, e.g., U.S. Patent Application Publication No. 2005/0226867 ).
  • the engineered Fc polypeptide comprises a C-terminal lysine (K) amino acid residue (e.g., human IgG1 heavy chain comprises a terminal lysine)
  • K C-terminal lysine
  • the lysine residue may be clipped resulting in a fusion protein lacking the C-terminal lysine residue.
  • the antibody or the Fc fusion protein comprising an engineered Fc polypeptide comprises a polypeptide where the terminal lysine otherwise present is not present.
  • the engineered Fc polypeptide of the disclosure comprises a substitution of the naturally occurring amino acid at position 297 wherein said substitution detectably reduces and/or abrogates glycosylation at position 297.
  • the engineered Fc polypeptide of the disclosure comprises a substitution of cysteine for asparagine at position 297 of the heavy chain of the antibody.
  • the disclosure provides antibodies lacking glycosylation at position 297 of the heavy chain of the antibody. In each of these, the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • an antibody therapeutic comprising an engineered Fc polypeptide of the disclosure can be modified with an appropriate sialylation profile for a particular application.
  • Methods for modulating the sialylation state of antibodies are presented in WO2007/005786 and WO2007/117505 .
  • the Fc region can be modified to increase the half-lives of proteins.
  • the increase in half-life allows for the reduction in amount of drug given to a patient as well as reducing the frequency of administration.
  • antibodies of the disclosure with increased half-lives may be generated by modifying (for example, substituting, deleting, or adding) amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor (see, for examples, PCT publication Nos. WO 97/34631 and WO 02/060919 , Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216 , Vaccaro et al., 2005, Nat. Biotechnol. 23(10):1283-1288 ).
  • the half-life of antibodies and fusion proteins of the disclosure may be increase by conjugation to a biopolymer (e.g., polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), hydroxyalkyl starch, XTEN (Amunix, Inc.), by techniques widely utilized in the art.
  • a biopolymer e.g., polyethylene glycol (PEG), albumin, hydroxyethyl starch (HES), hydroxyalkyl starch, XTEN (Amunix, Inc.
  • the engineered Fc polypeptides of antibodies of the disclosure comprise an increase in half-life of about 5 percent, about 10 percent, about 15 percent, about 20 percent, about 25 percent, about 30 percent, about 35 percent, about 40 percent, about 45 percent, about 50 percent, about 55 percent, about 60 percent, about 65 percent, about 70 percent, about 75 percent, about 80 percent, about 85 percent, about 90 percent, about 95 percent, about 100 percent, about 125 percent, about 150 percent or more as compared to a reference wild type unmodified Fc polypeptide.
  • the engineered Fc polypeptides of antibodies of the disclosure comprise an increase in half-life of about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold or more as compared to an unmodified reference Fc polypeptide.
  • the engineered Fc polypeptides of antibodies and Fc fusion proteins of the disclosure comprise a decrease in half-life.
  • the engineered Fc polypeptides of antibodies of the disclosure comprise a decrease in half-life of about 5 percent, about 10 percent, about 15 percent, about 20 percent, about 25 percent, about 30 percent, about 35 percent, about 40 percent, about 45 percent, about 50 percent, about 55 percent, about 60 percent, about 65 percent, about 70 percent, about 75 percent, about 80 percent, about 85 percent, about 90 percent, about 95 percent, about 100 percent, about 125 percent, about 150 percent or more as compared to a reference unmodified Fc polypeptide.
  • the engineered Fc polypeptides of antibodies of the disclosure comprise a decrease in half-life of about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold or more as compared to an unmodified reference Fc polypeptide.
  • the present disclosure provides Fc variants, wherein the engineered Fc polypeptide further comprises a non naturally occurring amino acid residue in addition to or other than, the substitutions disclosed above at one or more positions chosen from 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443 as numbered by the EU index as set forth in Kabat.
  • the engineered Fc polypeptide may comprise an additional non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Patents 5,624,821 , 6,277,375 , 6,737,056 , 7,217,797 , U.S. Patent Publication No. US2007/0135620 ; PCT Patent Publication Nos.
  • the present disclosure provides an engineered Fc variant antibody, wherein the engineered Fc polypeptide comprises at least one modification e.g., amino acid substitutions, amino acid insertions, amino acid deletions, amino acid additions) at one or more positions chosen from 234, 235, 237, and 331.
  • the non-naturally occurring amino acids are chosen from 234F, 235F, 235Y, and 331S.
  • the non-naturally occurring amino acids are chosen from 234A, 235A, and 237A.
  • the present disclosure provides an engineered Fc variant, wherein the Fc polypeptide comprises at least one non-naturally occurring amino acid at one or more positions chosen from 239, 330 and 332.
  • the non-naturally occurring amino acids are selected from the group chosen from 239D, 330L and 332E.
  • the present disclosure provides an engineered Fc variant antibody, wherein the Fc polypeptide comprises at least one non-naturally occurring amino acid at one or more positions chosen from 252, 254, and 256.
  • the non-naturally occurring amino acids are selected from the group chosen from 252Y, 254T and 256E (referred to as the "YTE modification"), as described in Dall'Acqua et al., 2006, J. Biol. Chem. 281:23514-23524 , and in U.S. Patent No. 7,083,784 .
  • the engineered Fc variant comprises an engineered Fc polypeptide comprising at least one amino acid substitution selected from a substitution at position 246, 249, 254, 265, 267, 270, 276, 278, 283, 284, 287, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 using the EU index as set forth in Kabat (supra), and further comprising at least one non-naturally occurring amino acid at one or more positions chosen from 428 and 434.
  • the additional amino acid substitutions comprise 428L and 434S as described
  • engineered variant antibodies of the disclosure may further comprise at least one or more non-naturally occurring cysteine amino acids in the 131-139 region of the CH1 domain of an antibody.
  • the engineered antibodies of the disclosure comprise at least one substitution at positions selected from: 131, 132, 133, 134, 135, 136, 137, 138, and 139 of the CH1 domain of an antibody, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • the engineered constant domain (Fc, C ⁇ , and C ⁇ ) polypeptide of the disclosure and an antibody, Fab, and F(ab') 2 comprising the engineered polypeptide may be produced by any method known in the art for the synthesis of antibodies, Fab and F(ab') 2 , in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988 ); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981 )).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a target antigen (either the full length protein or a domain thereof, e.g., the extracellular domain or the ligand binding domain) and once an immune response is detected, e.g., antibodies specific for the target antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution.
  • a target antigen either the full length protein or a domain thereof, e.g., the extracellular domain or the ligand binding domain
  • an immune response e.g., antibodies specific for the target antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well known techniques to any suitable myeloma cells
  • Hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the disclosure. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • monoclonal antibodies can be generated by culturing a hybridoma cell secreting an antibody of the disclosure wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with a target antigen with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind to a specific target antigen.
  • Antibody fragments which recognize specific target antigen epitopes may be generated by any technique known to those of skill in the art.
  • Fab and F(ab')2 fragments of the disclosure may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
  • the antibodies of the present disclosure can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues).
  • the DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB6 or pComb3 HSS).
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and M 13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to an epitope of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present disclosure include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50 ; Ames et al., 1995, J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • Techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in International Publication No.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 1 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions.
  • the vectors for expressing the VH or VL domains comprise an EF-1 ⁇ promoter, a secretion signal, a cloning site for the variable domain, constant domains, and a selection marker such as neomycin.
  • the VH and VL domains may also be cloned into one vector expressing the necessary constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • human or chimeric antibodies For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111 ; and International Publication Nos. WO 98/46645 , WO 98/50433 , WO 98/24893 , WO 98/16654 , WO 96/34096 , WO 96/33735 , and WO 91/10741 .
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the disclosure.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202 ; Oi et al, 1986, BioTechniques 4:214 ; Gillies et al., 1989, J. Immunol. Methods 125:191-202 ; and U.S. Patent Nos. 6,311,415 , 5,807,715 , 4,816,567 , and 4,816,397 .
  • Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting ( EP 0 239 400 ; International Publication No. WO 91/09967 ; and U.S. Patent Nos. 5,225,539 , 5,530,101 , and 5,585,089 ), veneering or resurfacing ( EP 0 592 106 ; EP 0 519 596 ; Padlan, 1991, Molecular Immunology 28(4/5):489-498 ; Studnicka et al., 1994, Protein Engineering 7:805 ; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969 ), and chain shuffling ( U.S. Patent No. 5,565,332 ).
  • Framework residues in the framework regions are typically substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No. 5,585,089 ; and Riechmann et al., 1988, Nature 332:323 ).
  • a humanized antibody is an antibody or its variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CH1, hinge, CH2, CH3, and CH4 (for IgA and IgM isotypes) regions of the heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4.
  • the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically human IgG1. Where such cytotoxic activity is not desirable, the constant domain may be of the human IgG2 class.
  • the humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. Such mutations, however, will not be extensive. Usually, at least 75 percent of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90 percent, or even greater than 95 percent.
  • FR parental framework region
  • Humanized antibodies can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 0 239 400 ; International Publication No. WO 91/09967 ; and U.S. Patent Nos. 5,225,539 , 5,530,101 , and 5,585,089 ), veneering or resurfacing (European Patent Nos. EP 0 592 106 and EP 0 519 596 ; Padlan, 1991, Molecular Immunology 28(4/5):489-498 ; Studnicka et al., 1994, Protein Engineering 7(6):805-814 ; and Roguska et al., 1994, Proc. Natl. Acad. Sci.
  • framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089 ; and Riechmann et al., 1988, Nature 332:323 ).
  • the antibodies of the disclosure can, in turn, be utilized to generate anti-idiotype antibodies using techniques well known to those skilled in the art. (See, e.g., Greenspan and Bona, 1989, FASEB J. 7:437-444 ; and Nissinoff, 1991, J. Immunol. 147:2429-2438 ).
  • the disclosure provides methods employing the use of polynucleotides comprising a nucleotide sequence encoding an antibody of the disclosure or a fragment thereof.
  • the antibodies of the disclosure comprise an Fc polypeptide comprising amino acid residue mutations (as numbered by the EU index as set forth in Kabat): M252Y/S254T/T256E or H433K/N434F/Y436H.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. Since the amino acid sequences of the antibodies are known, nucleotide sequences encoding these antibodies can be determined using methods well known in the art, i.e., nucleotide codons known to encode particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody or fragment thereof of the disclosure.
  • Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242 ), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known
  • the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al, 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998 , Current Protocols in Molecular Biology, John Wiley and Sons, NY ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
  • methods well known in the art for the manipulation of nucleotide sequences e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al, 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory,
  • one or more of the CDRs is inserted within framework regions using routine recombinant DNA techniques.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a listing of human framework regions).
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to 5T4, Her2 or VEGF.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen.
  • Such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibodies lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present disclosure and are within the skill of the art.
  • an engineered antibody including Fab and F(ab') 2 , comprising an engineered constant domain polypeptide of the disclosure, or a derivative, analog or fragment thereof, requires construction of an expression vector containing a polynucleotide that encodes the polypeptide.
  • the vector for the production of the antibody or engineered polypeptide comprising the same may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
  • the disclosure thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody of the disclosure, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036 ; and U.S. Patent No.
  • variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains, wherein the heavy chain comprises an engineered Fc region and/or the light chain comprises an engineered C ⁇ region of the disclosure.
  • the expression vector is transferred to a host cell by conventional techniques (transfection and transduction) and the host cells are then cultured by conventional techniques to produce an antibody of the disclosure.
  • the disclosure includes host cells containing a polynucleotide encoding an engineered Fc polypeptide, an engineered C ⁇ or C ⁇ polypeptide, or an antibody, Fab and F(ab') 2 comprising the same, or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single chain antibody of the disclosure, or a fusion protein comprising an engineered Fc polypeptide of the disclosure operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibodies and engineered polypeptides of the disclosure (see, e.g., U.S. Patent No. 5,807,715 ).
  • host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody of the disclosure in situ.
  • microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody, are used for the expression of engineered polypeptides and/or a recombinant antibody comprising the same.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector comprising the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies ( Foecking et al., 1986, Gene 45: 101 ; and Cockett et al., 1990, BioTechnology 8:2 ).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited to, the E. coli expression vector pUR278 ( Ruther et al., 1983, EMBO 12:1791 ), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pFN vectors ( Inouye and Inouye, 1985, Nucleic Acids Res.
  • PGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
  • GST glutathione 5-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into nonessential regions of the virus and placed under control of an AcNPV promoter.
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a nonessential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody in infected hosts (e.g., see Logan and Shenk, 1984, Proc. Natl. Acad.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:516-544 ).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, NS1 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030 and HsS78Bst cells.
  • the antibodies and fusion proteins comprising an engineered Fc polypeptide of the disclosure and/or an engineered C ⁇ or C ⁇ polypeptide of the disclosure are produced according to the methods disclosed in U.S. Patent No. 7,521,541 and U.S. Patent Application Publication No. 2009/0175865 .
  • cell lines which stably express the antibody may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody.
  • engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody.
  • a number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase ( Wigler et al, 1977, Cell 11:223 ), glutamine synthetase, hypoxanthine guanine phosphoribosyltransferase ( Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202 ), and adenine phosphoribosyltransferase ( Lowy et al., 1980, Cell 22:8-17 ) genes can be employed in tk - , gs - , hgprt - or aprt - cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate ( Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357 ; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527 ); gpt, which confers resistance to mycophenolic acid ( Mulligan and Berg, 1981, PNAS 78:2072 ); neo, which confers resistance to the aminoglycoside G-418 ( Wu and Wu, 1991, Biotherapy 3:87 ; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol.
  • the host cell may be co-transfected with two expression vectors of the disclosure, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain ( Proudfoot, 1986, Nature 322:52 ; and Kohler, 1980, PNAS 77:2197 ).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an engineered Fc polypeptide, or antibody, or antigen-binding portion thereof, or Fc fusion protein comprising the engineered Fc polypeptide, or an engineered C ⁇ or C ⁇ polypeptide may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the proteins of the present disclosure or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • the present disclosure encompasses the use of engineered antibody constant regions, e.g., Fc and/or C ⁇ , C ⁇ , or C ⁇ , and antibodies comprising the same (i.e., "engineered antibody"), which are recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous agent.
  • engineered Fab and F(ab')2 comprising an engineered constant domain region , e.g., Fc and/or C ⁇ , C ⁇ , or C ⁇ .
  • Antibody immunoconjugates are described in, among many others, Francisco et al., 2003, Blood 102:1458-1465 , Doronina et al., 2008, Bioconjugate Chem.
  • Suitable substances for attachment to the engineered antibodies of the disclosure include, but are not limited to, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus, a fluorophore, a chromophore, a dye, a toxin, a hapten, an enzyme, an antibody, an antibody fragment, a radioisotope, solid matrixes, semisolid matrixes and combinations thereof.
  • the disorder to be detected, treated, managed, or monitored is an autoimmune, inflammatory, infectious disease or cancer related disorder.
  • Methods for fusing or conjugating polypeptides to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603 , 5,622,929 , 5,359,046 , 5,349,053 , 5,447,851 , and 5,112,946 ; EP 0 307 434 ; EP 0 367 166 ; International Publication Nos.
  • DNA shuffling may be employed to alter the activities of engineered antibodies of the disclosure (e.g., antibodies with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793 ; 5,811,238 ; 5,830,721 ; 5,834,252 ; and 5,837,458 , and Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33 ; Harayama, 1998, Trends Biotechnol.
  • Antibodies or fragments thereof, or the encoded antibodies or fragments thereof may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • One or more portions of a polynucleotide encoding an antibody or antibody fragment may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous agents.
  • the engineered Fc regions, C ⁇ regions, and C ⁇ regions, or the antibodies of the disclosure comprising them are conjugated to a solid support.
  • Antibodies may be conjugated to a solid support as part of the screening and/or purification and/or manufacturing process.
  • antibodies of the disclosure may be conjugated to a solid support as part of a diagnostic method or composition.
  • a solid support suitable for use in the present disclosure is typically substantially insoluble in liquid phases. A large number of supports are available and are known to one of ordinary skill in the art.
  • solid supports include solid and semi-solid matrixes, such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or microplates), membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports
  • solid and semi-solid matrixes such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or microplates), membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports
  • the solid support may include a reactive functional group, including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc., for attaching the engineered antibodies of the disclosure.
  • a reactive functional group including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc.
  • engineered antibodies of the present disclosure or fragments or variants thereof are conjugated or fused to a marker sequence, such as a peptide, to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available. As described in Gentz et al, 1989, Proc. Natl. Acad. Sci. USA 86:821 , for instance, hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein ( Wilson et al., 1984, Cell 37:767 ) and the "flag" tag.
  • engineered antibodies of the present disclosure thereof are conjugated or fused to a diagnostic or detectable agent.
  • Such engineered antibodies can be useful for monitoring or prognosing the development or progression of a disorder (such as, but not limited to cancer) as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can accomplished by fusing or site-specifically conjugating the engineered antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin/biotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to, bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to, bismuth ( 213 Bi), carbon ( 14 C), chromium ( 51 Cr), cobal
  • engineered antibodies of the present disclosure are conjugated to a therapeutic agent such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters.
  • a therapeutic agent such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics ⁇ e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • Chemical toxins can also be taken from the group chosen from duocarmycin ( U.S. Patent Nos. 5,703,080 ; 4,923,990 ), methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.
  • chemotherapeutic agents also include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins ( U.S. Patent No. 4,675,187 ), Melphalan, and other related nitrogen mustards.
  • the cytotoxic agent is chosen from an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
  • the cytotoxic agent is paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretastatin, calicheamicin, maytansine, drug maytansinoid 1 (DM-1), an auristatin or other dolastatin derivatives, such as auristatin E or auristatin F, AEB, AEVB, AEFP, MMAD (monomethylauristatin D), MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), eleutherobin or netropsin.
  • DM-1 drug maytansinoid 1
  • auristatin or other dolastatin derivatives such as auristatin E or auristatin F, AEB, AEVB, AEFP,
  • auristatin E also known in the art as dolastatin-10, and its derivatives are described in U.S. Patent Application Publ. Nos. 2003/0083263 and 2005/0009751 ; International Patent Application No.: PCT/US02/13435 , U.S. Pat. Nos.
  • the cytotoxic agent is a novel cytotoxin disclosed in International Patent Application No. PCT/IB2012/056224 filed November 7, 2012 .
  • novel cytotoxic agents include, but are not limited to, 0101 (#54), 3377 (#115), and 8261 (#69) as described in the application which further discloses their synthesis.
  • the cytoxic agent is maytansine or maytansinoids, and derivatives thereof, wherein antibodies (full length or fragments) of the disclosure comprising an engineered constant region (C ⁇ , C ⁇ , C ⁇ , including an Fc), are site-specifically conjugated at the engineered amino acid substitution to one or more maytansinoid molecules.
  • Maytansinoids are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata ( U.S. Pat. No. 3,896,111 ), and additional maytansinoids were later isolated from other certain microbes, e.g., maytansinol and C-3 maytansinol esters ( U.S. Pat.
  • Immunoconjugates comprising maytansinoids non-specifically conjugated to an antibody and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020 , 5,416,064 and European Patent EP 0 425 235 ; Liu et al., 1996, Proc. Natl. Acad. Sci. USA 93:8618-8623 (immunoconjugates comprising DM1 non-specifically conjugated to mAb C242 targeting human colorectal cancer); Chari et al., 1992, Cancer Research 52: 127-131 (maytansinoid non-specifically conjugated to murine anti-colon cancer cell mAb A7 or murine mAb TA.1 anti-HER-2).
  • the present disclosure contemplates engineered antibodies site-specifically conjugated to maytansinoid agents for therapeutic treatment of certain cancers.
  • the drug is a maytansinoid. In a more specific embodiment, the drug is maytansine.
  • the cytotoxic or cytostatic agent is DM-1 (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res 52:127-131 ).
  • the cytotoxic agent of an engineered antibody conjugate of the disclosure is an anti-tubulin agent.
  • Anti-tubulin agents are a well established class of cancer therapy compounds. Examples of anti-tubulin agents include, but are not limited to, taxanes (e.g., Taxol® (paclitaxel), docetaxel), T67 (Tularik), vincas, and auristatins (e.g., auristatin E, AEB, AEVB, AEFP, MMAD, MMAE, MMAF, among others).
  • Antitubulin agents included in this class are also: vinca alkaloids, including vincristine and vinblastine, vindesine and vinorelbine; taxanes such as paclitaxel and docetaxel and baccatin derivatives, epithilone A and B, nocodazole, 5-Fluorouracil and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, dolastatins, discodermolide and eleutherobin
  • the cytotoxic agent is chosen from a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, and a dolastatin.
  • the cytotoxic agent is vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epithilone A, epithilone B, nocodazole, colchicine, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-1, an auristatin or other dolastatin derivatives, such as auristatin E or auristatin F, AEB, AEVB, AEFP, MMAD (monomethylauristatin D), MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), eleutherobin or netropsin.
  • the antibodies of the disclosure comprising an engineered Fc region may be conjugated or fused to other small molecule or protein toxins, such as, but not limited to abrin, brucine, cicutoxin, diphtheria toxin, batrachotoxin, botulism toxin, shiga toxin, endotoxin, Pseudomonas exotoxin, Pseudomonas endotoxin, tetanus toxin, pertussis toxin, anthrax toxin, cholera toxin, falcarinol, fumonisin BI, fumonisin B2, afla toxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, geldanamycin, gelonin, lotaustral
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAPS), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • the compounds used for conjugation to the antibody conjugates of the present disclosure can include conventional chemotherapeutics, such as doxorubicin, paclitaxel, carboplatin, melphalan, vinca alkaloids, methotrexate, mitomycin C, etoposide, and others.
  • chemotherapeutics such as doxorubicin, paclitaxel, carboplatin, melphalan, vinca alkaloids, methotrexate, mitomycin C, etoposide, and others.
  • potent agents such CC-1065 analogues, calichiamicin, maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can be linked to the antibodies at the enginnered conjugation site provided in the Fc region to provide potent immunoconjugates.
  • the cytotoxic or cytostatic agent is a dolastatin. In more specific embodiments, the dolastatin is of the auristatin class. In a specific embodiment of the disclosure, the cytotoxic or cytostatic agent is MMAD. In another specific embodiment of the disclosure, the cytotoxic or cytostatic agent is MMAE. In yet another specific embodiment of the disclosure, the cytotoxic or cytostatic agent is MMAF.
  • antibodies of the present disclosure or an engineered constant domain, or portion thereof are conjugated or fused to a therapeutic agent or drug moiety that modifies a given biological response.
  • Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-a, TNF-beta, AIM I (see, International Publication No.
  • a thrombotic agent or an anti-angiogenic agent e.g., angiostatin or endostatin
  • a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-4 (“IL-4"), interleukin-6 (“IL-6"), interleukin-7 (“IL-7”), interleukin-9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-15 (“IL-15”), interleukin-12 (“IL-12”), granulocyte macrophage colony stimulating factor (“GM- CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or a growth factor (e.g., growth hormone (“GH”)), and a receptor, or ligand binding portion thereof, of any of the preceding molecules.
  • a lymphokine e.g., interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-4
  • engineered antibodies of the present disclosure are specifically conjugated to a polypeptide that comprises poly-arginine or poly-lysine residues.
  • said polypeptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid residues.
  • the poly-arginine polypeptide may comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more arginine residues.
  • the poly-lysine polypeptide may comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more lysine residues.
  • the polypeptide may comprise any combination of arginine and lysine residues.
  • engineered antibodies of the present disclosure are conjugated to a therapeutic agent such as radioactive materials or macrocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials).
  • the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • DOTA 1,4,7,10-tetraazacyclododecane-N,N',N",N"-tetraacetic acid
  • linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4:2483-90 ; Peterson et al, 1999, Bioconjug. Chem. 10:553 ; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50 .
  • engineered antibodies of the present disclosure are conjugated to a nucleic acid.
  • the nucleic acid may be selected from DNA, RNA, short interfering RNA (siRNA), microRNA, hairpin or nucleic acid mimetics such as peptide nucleic acid.
  • the conjugated nucleic acid is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 at least 100, at least 200, at least 500, at least 1000, at least 5000 or more base pairs.
  • the conjugated nucleic acid is single stranded. In alternative embodiments, the conjugated nucleic acid is double stranded.
  • Moieties can be conjugated to antibodies by any method known in the art, including, but not limited to aldehyde/Schiff linkage, sulfhydryl linkage, acid-labile linkage, cis-aconityl linkage, hydrazone linkage, enzymatically degradable linkage (see generally Garnett, 2002, Adv. Drug Deliv. Rev. 53:171-216 ). Additional techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R.
  • Two exemplary approaches may be taken to minimize drug activity outside the cells that are targeted by the antibody conjugates of the disclosure: first, an antibody that binds to a cell membrane receptor but not soluble receptor may be used, so that the drug, including drug produced by the actions of the prodrug converting enzyme, is concentrated at the cell surface of the cells, such as an activated lymphocyte; second, the drugs are conjugated in a manner that would reduce their activity unless they are hydrolyzed or cleaved off the antibody.
  • Such methods would employ attaching the drug to the antibodies with linkers that are sensitive to the environment at the cell surface of the activated lymphocyte (e.g., the activity of a protease that is present at the cell surface of the activated lymphocyte) or to the environment inside the activated lymphocyte the conjugate encounters when it is taken up by the activated lymphocyte (e.g., in the endosomal or, for example by virtue of pH sensitivity or protease sensitivity, in the lysosomal environment).
  • linkers that can be used in the present disclosure are disclosed in U.S. Patent Application Publication Nos. 2005/0123536 , 2005/0180972 , 2005/0113308 , 2004/0157782 , and U.S. Patent No. 6,884,869 .
  • the linker is an acid-labile hydrazone or hydrazide group that is hydrolyzed in the lysosome (see, e.g., U.S. Pat. No. 5,622,929 ).
  • drugs can be conjugated to antibodies through other acid-labile linkers, such as cis-aconitic amides, orthoesters, acetals and ketals ( Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123 ; Neville et al., 1989, Biol. Chem. 264: 14653-14661 ).
  • Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5, the approximate pH of the lysosome.
  • drugs are attached to the antibodies of the disclosure at an engineered reactive site using peptide spacers that are cleaved by intracellular proteases.
  • Target enzymes include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells ( Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123 ).
  • the advantage of using intracellular proteolytic drug release is that the drug is highly attenuated when conjugated and the serum stabilities of the conjugates can be extraordinarily high.
  • the cathepsin B sensitive dipeptide linker is valine-citrulline (referred to herein as “val-cit” or “ValCit”) as described in, e.g., Gerber et al., 2009, Blood 113(18):4352-4361 .
  • the engineered Fc polypeptide and/or the engineered C ⁇ polypeptide of an engineered antibody is site-specifically conjugated to a cleavable linker, e.g., val-cit, which is conjugated to an auristatin, such as, but not limited to, val-cit-MMAD, val-cit-MMAE, and val-cit-MMAF, among many others.
  • a cleavable linker e.g., val-cit
  • an auristatin such as, but not limited to, val-cit-MMAD, val-cit-MMAE, and val-cit-MMAF, among many others.
  • the engineered Fc polypeptide and/or the engineered C ⁇ polypeptide of the disclosure, or an engineered antibody comprising the polypeptide is site-specifically conjugated to a noncleavable linker, e.g., maleimidocaproyl (mc or mal-c), which is conjugated to an auristatin, such as, but not limited to, mc-MMAD, mc-MMAE, and mc-MMAF, among many others.
  • a noncleavable linker e.g., maleimidocaproyl (mc or mal-c)
  • auristatin such as, but not limited to, mc-MMAD, mc-MMAE, and mc-MMAF, among many others.
  • maleimido can be represented by "mal”.
  • noncleavable linkers may be used to site-specifically conjugate a cytotoxic or cytostatic agent to an engineered Fc polypeptide and/or an engineered C ⁇ polypeptide or an engineered antibody comprising an engineered polypeptide of the disclosure.
  • Noncleavable linkers include, but are not limited to, maleimidocaproyl (mc) linkers such as those described in Lee et al., 2009, J. Natl. Cancer Inst. 2009 101:1193-1205 (conjugating MMAF with an anti-EphA2 antibody using a maleimidocaproyl linker).
  • the linker is a malonate linker ( Johnson et al., 1995, Anticancer Res. 15:1387-1393 ), a maleimidobeiizoyl linker ( Lau et al., 1995, Bioorg. Med. Chem. 3(10):1299-1304 ), or a 3'-N-amide analog ( Lau et al, 1995, Bioorg. Med. Chem. 3(10):1305-1312 ).
  • Linking chemistry employing a maleimide group and a spacer (such as polyethylene glycol (PEG) or the like) is suitable for cysteine, lysine, selenocysteine, and selenomethaionine substitutions.
  • a spacer such as polyethylene glycol (PEG) or the like
  • cysteine lysine
  • selenocysteine selenocysteine
  • selenomethaionine substitutions one may use a spacer coupled with a metal (such as copper, zinc, iron, nickel, etc.) for conjugation.
  • tyrosine one may conjugate to a functional group present in a sugar or other hydroxyl compound. Details of these and other suitable conjugation techniques are known those of ordinary skill in the art and can be found in, for example, Bioconjugate Techniques, 2nd ed., by Greg T. Hermanson, Academic Press (2008).
  • the noncleavable linker is maleimidocaproyl linked to a spacer such as, but not limited to, PEG2-C2, PEG3-C2 and PEG6-C2 to form a linker-spacer moiety mc-PEG2-C2, mc-PEG3-C2, and mc-PEG6-C2, among others.
  • a spacer such as, but not limited to, PEG2-C2, PEG3-C2 and PEG6-C2 to form a linker-spacer moiety mc-PEG2-C2, mc-PEG3-C2, and mc-PEG6-C2, among others.
  • the linker is a cleavable linker such as, but not limited to, valine-citrulline which is susceptible to cathepsin B cleavage, which is conjugated to a spacer moiety such as, but not limited to, PEG2-C2, PEG3-C2, and PEG6-C2 to form a linker-spacer moiety including val-cit-PEG2-C2, val-cit-PEG3-C2 and val-cit-PEG6-C2, among others.
  • a cleavable linker such as, but not limited to, valine-citrulline which is susceptible to cathepsin B cleavage, which is conjugated to a spacer moiety such as, but not limited to, PEG2-C2, PEG3-C2, and PEG6-C2 to form a linker-spacer moiety including val-cit-PEG2-C2, val-cit-PEG3-C2 and val-cit-PEG6
  • the cleavable linker-spacer moiety is conjugated to an auristatin, including, but not limited to, MMAD, MMAE, MMAF, 0101, 3377, and 8261.
  • the linker-spacer-auristatin encompasses mc-val-cit-PABC-MMAD (vc-MMAD), mc-val-cit-PABC-MMAE (vc-MMAE) and mc-val-cit-PABC-MMAF (vc-MMAF).
  • the linker-spacer-auristatin encompasses mc-val-cit-PABC-PEG2-C2-MMAD (vc-PEG-C2-MMAD), mc-val-cit-PABC-PEG3-C2-MMAD (vc-PEG3-C2-MMAD), mc-val-cit-PABC-PEG6-C2-MMAD (vc-PEG6-C2-MMAD), mc-val-cit-PABC-PEG2-C2-MMAE (vc-PEG2-C2-MMAE), mc-val-cit-PABC-PEG3-C2-MMAE (vc-PEG3-C2-MMAE), mc-val-cit-PABC-PEG6-C2-MMAE (vc-PEG6-C2-MMAE), mc-val-cit-PABC-PEG2-C2-MMAE (vc-PEG6-C2-MMAE), mc-val-cit-PABC-PEG
  • the noncleavable linker-spacer moiety is further conjugated to an auristatin, including, but not limited to, MMAD, MMAE, MMAF, 0101, 3377, and 8261.
  • the linker-spacer-auristatin encompasses mc-PEG2-C2-MMAD, mc-PEG3-C2-MMAD, mc-PEG6-C2-MMAD, mc-PEG2-C2-MMAE, mc-PEG3-C2-MMAE, mc-PEG6-C2-MMAE, mc-PEG2-C2-MMAF, mc-PEG3-C2-MMAF, and mc-PEG6-C2-MMAF, among others.
  • a cytotoxic agent is conjugated to an engineered Fc polypeptide via a linker.
  • a cytotoxic agent is conjugated to an engineered antibody constant domain polypeptide, or portion thereof, via a linker such as, but not limited to, the linkers described herein or known in the art, and the cytotoxic agent is an auristatin, a maytansinoid and a calicheamicin, among others.
  • an engineered antibody constant domain polypeptide, or portion thereof, comprising an introduced cysteine is conjugated via a linker and cytotoxic agent combination including, but not limited to, maleimidocaproyl-monomethyl auristatin D (mcMMAD), maleimidocaproyl-monomethyl auristatin E (mcMMAE), maleimidocaproyl-monomethyl auristatin F (mcMMAF), maleimidocaproyl-0101 (mc0101), maleimidocaproyl-3377 (mc3377), maleimidocaproyl-8261 (mc8261), valine-citrulline-monomethyl auristatin D (vcMMAD), valine-citrulline-monomethyl auristatin E (vcMMAE), valine-citrulline-monomethyl auristatin F (vcMMAF), valine-citrulline-0101 (vc0101), valine-citrulline
  • a cytotoxic agent is conjugated to an engineered C ⁇ polypeptide via a linker.
  • a cytotoxic agent is conjugated to an engineered antibody constant domain (e.g., C ⁇ , C ⁇ , and C ⁇ ) polypeptide, or portion thereof, via a linker, to a cytotoxic agent, wherein the linker and the cytotoxic agent are selected from the group consisting of maleimidocaproyl-monomethyl auristatin D (mcMMAD), maleimidocaproyl-monomethyl auristatin E (mcMMAE), maleimidocaproyl-monomethyl auristatin F (mcMMAF), maleimidocaproyl-0101 (mc0101), maleimidocaproyl-3377 (mc3377) maleimidocaproyl-8261 (mc8261), valine-citrulline-monomethyl auristatin D (vcMMAD), valine-citrulline-monomethyl auristatin E (vcMMAE), valine-citrulline-monomethyl auristatin
  • a cytotoxic agent is conjugated to an engineered C ⁇ polypeptide via a linker such as, but not limited to, the linkers described herein or known in the art, and the cytotoxic agent is an auristatin, a maytansinoid and a calicheamicin, among others.
  • an engineered C ⁇ or C ⁇ polypeptide comprising an introduced cysteine is conjugated via a linker and cytotoxic agent combination including, but not limited to, maleimidocaproyl-monomethyl auristatin D (mcMMAD), maleimidocaproyl-monomethyl auristatin E (mcMMAE), maleimidocaproyl-monomethyl auristatin F (mcMMAF), maleimidocaproyl-0101 (mc0101), maleimidocaproyl-3377 (mc3377), maleimidocaproyl-8261 (mc8261) valine-citrulline-monomethyl auristatin D (vcMMAD), valine-citrulline-monomethyl auristatin E (vcMMAE), valine-citrulline-monomethyl auristatin F (vcMMAF), valine-citrulline-0101 (vc0101), valine-citrulline-3377
  • engineered antibody conjugates are generally made by conjugating a compound or a drug to an engineered antibody, or an engineered Fc polypeptide and/or engineered C ⁇ polypeptide, through a linker.
  • Any linker that is known in the art may be used in the conjugates of the present disclosure, e.g., bifunctional agents (such as dialdehydes or imidoesters) or branched hydrazone linkers (see, e.g., U.S. Pat. No. 5,824,805 .
  • the linker region between the conjugate moiety and the engineered antibody/engineered Fc/C ⁇ polypeptide moiety is cleavable under certain conditions, wherein cleavage or hydrolysis of the linker releases the drug moiety from the antibody/engineered Fc/C ⁇ moiety.
  • the linker is sensitive to cleavage or hydrolysis under intracellular conditions.
  • the linker region between the conjugate moiety and the engineered antibody moiety is cleavable if the pH changes by a certain value or exceeds a certain value.
  • the linker is cleavable in the milieu of the lysosome, e.g., under acidic conditions (i.e., a pH of around 5-5.5 or less).
  • the linker is a peptidyl linker that is cleaved by a peptidase or protease enzyme, including but not limited to a lysosomal protease enzyme, a membrane-associated protease, an intracellular protease, or an endosomal protease.
  • the linker is at least two amino acids long, more typically at least three amino acids long.
  • a peptidyl linker that is cleavable by cathepsin-B e.g., a Val-Cit linker, a Gly-Phe-Leu-Gly linker, among others
  • a thiol-dependent protease that is highly expressed in cancerous tissue.
  • Other such linkers are described, e.g., in U.S. Pat. No. 6,214,345 .
  • the linker by which the engineered antibody and compound of an antibody conjugate of the disclosure are conjugated promotes cellular internalization.
  • the linker-drug moiety promotes cellular internalization.
  • the linker is chosen such that the structure of the entire antibody conjugate promotes cellular internalization.
  • the linker is a thioether linker (see, e.g., U.S. Pat. No. 5,622,929 ).
  • the linker is a hydrazone linker (see, e.g., U.S. Pat. Nos. 5,122,368 , and 5,824,805 ).
  • the linker is a disulfide linker.
  • disulfide linkers are known in the art, including but not limited to those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldi-thio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3-(2-pyridyldi-thio)propionate
  • SPDB N-succinimidyl-3-(2-pyridyldithio)buty
  • Elimination of amine-containing drugs that are substituted at the a-position of glycine are also examples of self-immolative spacer strategies that can be applied to the antibody-linker-drug conjugates of the disclosure.
  • the linker unit of an antibody conjugate links the cytotoxic or cytostatic agent (drug; -D) and the antibody (-Ab).
  • the linker unit has the general formula: -T a -W w -Y y - wherein:
  • Useful functional groups that can be present on an antibody, either naturally or via chemical manipulation include, but are not limited to, sulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl.
  • Other methods of introducing sulfhydryl groups involve the reduction of the intramolecular disulfide bonds of an antibody.
  • sulfhydryl groups can be generated by reaction of an amino group of an engineered lysine moiety of an antibody (which has been introduced) with 2-iminothiolane (Traut's reagent) or other sulfhydryl generating reagents.
  • the amino acid unit (-W-) links the stretcher unit (-T-) to the Spacer unit (-Y-) if the Spacer unit is present, and links the stretcher unit to the cytotoxic or cytostatic agent (drug; D) if the spacer unit is absent.
  • -W W - is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
  • the amino acid unit of the linker unit can be enzymatically cleaved by an enzyme including, but not limited to, a tumor-associated protease, cathepsin B, cathepsin D, plasmin, and the like, to liberate the drug (-D) which is protonated in vivo upon release to provide a cytotoxic drug (D).
  • the amino acid unit is a phenylalanine-lysine dipeptide (phe-lys or FK linker). In another embodiment, the amino acid unit is a valine-citrulline dipeptide (val-cit).
  • Spacer units are of two general types: self-immolative and non self-immolative.
  • a non self-immolative spacer unit is one in which part or all of the spacer unit remains bound to the drug unit after enzymatic cleavage of an amino acid unit from the antibody-linker-drug conjugate or the drug-linker compound.
  • Examples of a non self-immolative spacer unit include, but are not limited to a (glycine-glycine) spacer unit and a glycine spacer unit.
  • an antibody-linker-drug conjugate of the disclosure containing a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage via a tumor-cell associated-protease, a cancer-cell-associated protease or a lymphocyte-associated protease, a glycine-glycine-drug moiety or a glycine-drug moiety is cleaved from Ab-T-W W -.
  • an independent hydrolysis reaction should take place within the target cell to cleave the glycine-drug unit bond.
  • self-immolative spacers include, but are not limited to, para-aminobenzyloxycarbonyl (PABC) and aromatic compounds that are electronically equivalent to the PABC group such a 2-aminoimidazol-5-methanol derivatives (see Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237 for examples) and ortho- or para-aminobenzylacetals.
  • Spacers can be used that undergo facile cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides ( Rodrigues et al., Chemistry, Biology, 1995, 2, 223 ), appropriately substituted ring systems ( Storm, et al., J. Amer. Chem. Soc, 1972, 94, 5815 ) and 2-aminophenylpropionic acid amides ( Amsberry, et al., J. Org. Chem., 1990, 55, 5867 ).
  • PABC para-
  • Heterologous molecules such as those described herein may be efficiently conjugated to engineered antibodies comprising an engineered Fc region and/or an engineered C ⁇ region and/or an engineered C ⁇ region of the disclosure through the reactive groups the engineered amino acid residues provide.
  • the disclosure provides methods for efficiently conjugating heterologous molecules to cysteine engineered antibodies.
  • the conjugation of a heterologous molecule may occur at a reactive group provided by at least one engineered residue selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the Fc region or antibody comprising the Fc polypeptide, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • the reactive group is a thiol
  • the conjugation of a heterologous molecule may occur at a thiol group provided by at least one engineered cysteine residue selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the Fc polypeptide or an antibody comprising the Fc polypeptide, wherein the numbering system of the constant region is that of the EU index as set forth in Kabat.
  • the conjugation of a heterologous molecule may occur at a reactive group provided by at least one engineered residue selected from the positions 111, 149, 183, 188, 207, and 210, of the engineered C ⁇ polypeptide or an engineered antibody comprising the engineered C ⁇ polypeptide, wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat.
  • the reactive group is a thiol
  • the conjugation of a heterologous molecule may occur at a thiol group provided by at least one engineered cysteine residue selected from the positions 111, 149, 183, 188, 207, and 210, of the engineered C ⁇ polypeptide or an engineered antibody comprising the engineered C ⁇ polypeptide, wherein the numbering system of the constant region is that of the Kabat numbering index as set forth in Kabat.
  • the engineering of non-naturally occurring cysteine residues into antibodies may alter the disulfide pairing of the heavy and light chains such that a naturally occurring cysteine residue which was part of a disulfide bond is liberated and presents a thiol group capable of conjugation.
  • the method comprises the efficient conjugation of a heterologous molecule to a cysteine engineered antibody at a thiol group provided by at least one engineered cysteine residue selected from the positions 246, 249, 265, 267, 270, 276, 278, 283, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 376, 378, 380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 of the Fc region of an antibody.
  • the engineering of non-naturally occurring cysteine residues into antibodies may alter the disulfide pairing of the heavy and light chains such that a naturally occurring cysteine residue which was part of a disulfide bond is liberated and presents a thiol group capable of conjugation.
  • the method comprises the efficient conjugation of a heterologous molecule to a cysteine engineered antibody at a thiol group provided by at least one engineered cysteine residue selected from the positions 111, 149, 183, 188, 207, and 210, of the C ⁇ region of an antibody.
  • the presence of free thiol groups in antibodies may be determined by various art accepted techniques, such as those described herein infra.
  • the efficiency of conjugation of a heterologous molecule to an antibody may be determined by assessing the presence of free thiols remaining after the conjugation reaction.
  • the disclosure provides a method of efficiently conjugating a heterologous molecule to a cysteine engineered antibody.
  • the conjugation efficiency is at least 5 percent, at least 20 percent, at least 50 percent, at least 80 percent, at least 90 percent, at least 95 percent, at least 98 percent, at least 99 percent, or more as measured by the level of free thiol groups remaining after the conjugation reaction.
  • the disclosure provides a method of conjugating a heterologous molecule to an engineered antibody, including a Fab or F(ab')2, or engineered Fc region and/or C ⁇ region or C ⁇ of the antibody wherein the antibody or Fc, C ⁇ and/or C ⁇ region comprises at least one amino acid substitution, such that 2 or more reactive groups are formed.
  • the method comprises an engineered Fc polypeptide, an engineered C ⁇ polypeptide, an engineered C ⁇ polypeptide, or engineered antibody comprising at least one amino acid substitution, such that at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or more newly-introduced reactive groups are formed.
  • at least one of the substitutions is with a cysteine, and the reactive groups are thiol groups.
  • Engineered constant regions (Fc, C ⁇ , and C ⁇ ), and antibodies comprising them of the disclosure capable of conjugation may contain cysteine residues that comprise sulfhydryl groups that are blocked or capped. Such caps include proteins, peptides, ions and other materials that interact with the sulfhydryl group and prevent or inhibit conjugate formation. Antibodies of the disclosure may require uncapping prior to a conjugation reaction.
  • engineered constant regions (Fc, C ⁇ , and C ⁇ ), and engineered antibodies of the disclosure comprising the polypeptides are uncapped and display a sulfhydryl group capable of conjugation.
  • antibodies of the disclosure are subjected to an uncapping reaction that results in minimal disruption or rearrangement of the naturally occurring disulfide bonds.
  • the level of naturally occurring disulfide bond disruption may range from about 30% to an undetectable level compared with the level of disruption in the untreated polypeptide.
  • antibodies of the disclosure are subjected to an uncapping reaction as presented in International Patent Publication Nos. WO 2008/141044 , WO 2009/092011 , and WO 2010/1411902 .
  • engineered antibodies of the disclosure may be subjected to conjugation reactions wherein the antibody to be conjugated is present at a concentration of at least 1 mg/ml, at least 2 mg/ml, at least 3 mg/ml, at least 4 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 13 mg/ml, at least 14 mg/ml, at least 15 mg/ml, at least 16 mg/ml or higher.
  • the engineered antibody conjugates can be used for diagnostic imaging.
  • the engineered antibody conjugate can be a radiolabeled monoclonal antibody. See, for example, Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy, Plenum Press (1988 ); Chase, “Medical Applications of Radioisotopes,” in Remington's Pharmaceutical Sciences, 18th Edition, Gennaro et al. (eds.), Mack Publishing Co., pp. 624-652 (1990 ); and Brown, “Clinical Use of Monoclonal Antibodies,” in Biotechnology and Pharmacy, Pezzuto et al. (eds.), Chapman and Hall, pp. 227-249 (1993 ).
  • This technique also known as immunoscintigraphy, uses a gamma camera to detect the location of gamma-emitting radioisotopes conjugated to monoclonal antibodies. Diagnostic imaging can be used to diagnose cancer, autoimmune disease, infectious disease and/or cardiovascular disease. (See, e.g., Brown, supra.)
  • the engineered antibody conjugates can be used to diagnose cardiovascular disease.
  • engineered antibody conjugates comprising anti-myosin antibody fragments can be used for imaging myocardial necrosis associated with acute myocardial infarction.
  • engineered antibody conjugates comprising antibody fragments that bind to platelets or fibrin can be used for imaging deep-vein thrombosis.
  • engineered antibody conjugates comprising antibody fragments that bind to activated platelets can be used for imaging atherosclerotic plaque.
  • Engineered antibody conjugates can also be used in the diagnosis of infectious diseases.
  • engineered antibody conjugates comprising antibody fragments that bind specific bacterial antigens can be used to localize abscesses.
  • engineered antibody conjugates comprising antibody fragments that bind granulocytes and inflammatory leukocytes can be used to localize sites of bacterial infection.
  • tumor markers include carcinoembryonic antigen, alpha-fetoprotein, oncogene products, tumor-associated cell surface antigens, and necrosis-associated intracellular antigens, as well as the tumor-associated antigens and tumor-specific antigens discussed infra.
  • monoclonal antibody imaging can be used to monitor therapeutic responses, detect recurrences of a disease, and guide subsequent clinical decisions.
  • radioisotopes may be bound to antibody fragments either directly or indirectly by using an intermediary functional group.
  • intermediary functional groups include, for example, DTPA (diethylenetriaminepentaacetic acid) and EDTA (ethylene diamine tetraacetic acid).
  • the radiation dose delivered to the patient is typically maintained at as low a level as possible. This may be accomplished through the choice of isotope for the best combination of minimum half-life, minimum retention in the body, and minimum quantity of isotope which will permit detection and accurate measurement.
  • Examples of radioisotopes which can be bound to antibodies and are appropriate for diagnostic imaging include 99 mTc and 111 In.
  • antibody fragments particularly Fab and Fab', provide suitable tumor/background ratios. (See, e.g., Brown, supra.)
  • the engineered antibody conjugates also can be labeled with paramagnetic ions for purposes of in vivo diagnosis.
  • Elements which are particularly useful for Magnetic Resonance Imaging include Gd, Mn, Dy, and Fe ions.
  • the engineered antibody conjugates can also detect the presence of particular antigens in vitro.
  • the engineered antibody conjugates may be utilized in liquid phase or bound to a solid-phase carrier.
  • an intact antibody, or antigen-binding fragment thereof can be attached to a polymer, such as aminodextran, in order to link the antibody component to an insoluble support such as a polymer-coated bead, plate, or tube.
  • the engineered antibody conjugates can be used to detect the presence of particular antigens in tissue sections prepared from a histological specimen.
  • in situ detection can be accomplished, for example, by applying a detectably-labeled immunoconjugate to the tissue sections.
  • In situ detection can be used to determine the presence of a particular antigen and to determine the distribution of the antigen in the examined tissue.
  • General techniques of in situ detection are well known to those of ordinary skill. (See, e.g., Ponder, "Cell Marking Techniques and Their Application,” in Mammalian Development: A Practical Approach, Monk (ed.), IRL Press, pp. 115-138 (1987 ); Coligan et al., supra.)
  • Detectable labels such as enzymes, fluorescent compounds, electron transfer agents, and the like can be linked to a carrier by conventional methods well known to the art.
  • These labeled carriers and the engineered antibody conjugates prepared from them can be used for in vitro immunoassays and for in situ detection, much as an antibody conjugate can be prepared by direct attachment of the labels to antibody.
  • the loading of the engineered antibody conjugates with a plurality of labels can increase the sensitivity of immunoassays or histological procedures, where only a low extent of binding of the antibody, or antibody fragment, to target antigen is achieved.
  • Engineered antibody conjugates can be used to treat viral and bacterial infectious diseases, cardiovascular disease, autoimmune disease, and cancer.
  • the objective of such therapy is to deliver cytotoxic or cytostatic doses of an active agent (e.g., radioactivity, a toxin, or a drug) to target cells, while minimizing exposure to non-target tissues.
  • an active agent e.g., radioactivity, a toxin, or a drug
  • a radioisotope can be attached to an intact antibody, or antigen-binding fragment thereof, directly or indirectly, via a chelating agent.
  • a chelating agent for example, 67 Cu can be conjugated to an antibody component using the chelating agent, p-bromo-acetamidobenzyl-tetraethylaminetetraacetic acid (TETA).
  • TETA p-bromo-acetamidobenzyl-tetraethylaminetetraacetic acid
  • engineered antibody conjugates can be prepared in which the therapeutic agent is a toxin or drug.
  • Useful toxins for the preparation of such engineered antibody conjugates include ricin, abrin, pokeweed antiviral protein, gelonin, diphtherin toxin, and Pseudomonas endotoxin.
  • chemotherapeutic drugs for the preparation of immunoconjugates include auristatin, dolastatin, MMAE, MMAF, AFP, AEB, doxorubicin, daunorubicin, methotrexate, melphalan, chlorambucil, vinca alkaloids, 5-fluorouridine, mitomycin-C, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustards, cytoxan, etoposide, BCNU, irinotecan, camptothecins, bleomycin, idarubicin, dactinomycin, plicamycin, mitoxantrone, asparaginas
  • chelators such as DTPA, to which detectable labels such as fluorescent molecules or cytotoxic agents such as heavy metals or radionuclides can be complexed; and toxins such as Pseudomonas exotoxin, and the like.
  • the diagnostic, preventative or therapeutic agent is auristatin E (also known in the art as dolastatin-10) or a derivative thereof as well as pharmaceutically salts or solvates thereof.
  • the auristatin E derivative is, e.g., an ester formed between auristatin E and a keto acid.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatin derivatives include AFP, MMAF, and MMAE.
  • the anti-cancer agent includes, but is not limited to, a drug listed in below: methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinote
  • suitable chemotherapeutics include, but are not limited to, alkylating agents : nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil); nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU)); alkylsulphonates ( e.g., busulfan, treosulfan); triazenes ( e.g., dacarbazine); Platinum containing compounds ( e.g., cisplatin, carboplatin, aroplatin, oxaliplatin); Plant Alkaloids : Vinca alkaloids ( e.g., vincristine, vinblastine, vindesine, vinorelbine); Taxoids ( e.g ., paclitaxel, docetaxel; DNA Topoisomerase Inhibitors : epipodophyllins (e.g.
  • Additional anti-cancer agents that may be used in the methods of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amifostine trihydrate; aminoglutethimide; amsacrine; anastrozole; anthramycin; arsenic trioxide; asparaginase; asperlin; azacitidine; azetepa; azotomycin; Bacillus Calmette-Guerin; batimastat; benzodepa; bevacizumab; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; bortezomib; brequinar sodium; bropirimine; busulfan; cactin
  • anti-cancer drugs that can be used include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL
  • the diagnostic, preventative or therapeutic agent is not a radioisotope.
  • an engineered antibody conjugate can be used to treat one of the following particular types of cancer: It is contemplated that the engineered antibody conjugates of the present disclosure may be used to treat various diseases or disorders, e.g. those characterized by the overexpression of a tumor antigen. Exemplary conditions or hyperproliferative disorders include benign or malignant tumors, leukemia and lymphoid malignancies. Others include neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, endothelial, and stromal malignancies.
  • cancers or hyperproliferative disorders include: cancers of the head, neck, eye, mouth, throat, esophagus, chest, skin, bone, lung, colon, rectum, colorectal, stomach, spleen, kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma, endometrial, prostate, breast, ovaries, testicles, thyroid, blood, lymph nodes, kidney, liver, pancreas, brain, or central nervous system.
  • cancers that can be prevented, managed, treated or ameliorated in accordance with the methods of the disclosure include, but are not limited to, cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, and brain.
  • Additional cancers include, but are not limited to, the following: leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblasts, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Walden
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al, 1985, Medicine, 2d Ed., J. B.
  • cancers caused by aberrations in apoptosis can also be treated by the methods and compositions of the disclosure.
  • Such cancers may include, but not be limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.
  • the engineered antibody conjugates of the disclosure, and the engineered Fc polypeptides and engineered C ⁇ polypeptides, and compositions comprising the same are useful for many purposes, for example, as therapeutics against a wide range of chronic and acute diseases and disorders including, but not limited to, autoimmune and/or inflammatory disorders, which include Sjogren's syndrome, rheumatoid arthritis, lupus psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g.,
  • autoimmune and/or inflammatory disorders include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome, psoriasis, atherosclerosis, diabetic and other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, and chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, reperfusion injury, septicemia, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock,
  • inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections.
  • COPD chronic obstructive pulmonary disease
  • the compositions and methods of the disclosure can be used with one or more conventional therapies that are used to prevent, manage or treat the above diseases.
  • the disclosure also provides methods of using the engineered antibody conjugates of the disclosure, and the engineered Fc polypeptides and engineered C ⁇ polypeptides of the disclosure, to inactivate various infectious agents such as viruses, fungi, eukaryotic microbes, and bacteria.
  • the compositions of the disclosure may be used to inactivate RSV, hMPV, PIV, or influenza viruses.
  • compositions of the disclosure may be used to inactivate fungal pathogens, such as, but not limited to members Naegleria, Aspergillus, Blastomyces, Histoplasma, Candida or Tinea genera.
  • compositions of the disclosure may be used to inactivate eukaryotic microbes, such as, but not limited to members of Giardia, Toxoplasma, Plasmodium, Trypanosoma, and Entamoeba genera.
  • compositions of the disclosure may be used to inactivate bacterial pathogens, such as but not limited to members of Staphylococcus, Streptococcus, Pseudomonas, Clostridium, Borrelia, Vibro and Neiserria genera.
  • compositions of the disclosure are useful for many purposes, for example, as therapeutics against a wide range of chronic and acute diseases and disorders including, but not limited to, infectious disease, including viral, bacterial and fungal diseases.
  • viral pathogens include but are not limited to: adenovirdiae (e.g., mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae ⁇ e.g., levivirus, enterobacteria phase MS2, allolevirus), poxviridae (e.g., chordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxvirinae), papovaviridae (e.g., polyo
  • human immunodeficiency virus 1 and human immunodeficiency virus T spumavirus
  • flaviviridae e.g., hepatitis C virus
  • hepadnaviridae e.g., hepatitis B virus
  • togaviridae e.g., alphavirus (e.g., Sindbis virus) and rubivirus (e.g., rubella virus)
  • rhabdoviridae e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus
  • arenaviridae e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, and lassa virus
  • coronaviridae e.g., coronavirus and torovirus
  • bacterial pathogens include but are not limited to: but not limited to, the Aquaspirillum family, Azospirillum family, Azotobacteraceae family, Bacteroidaceae family, Bartonella species, Bdellovibrio family, Campylobacter species, Chlamydia species (e.g., Chlamydia pneumoniae), Clostridium, Enterobacteriaceae family (e.g., Citrobacter species, Edwardsiella, Enterobacter aerogenes, Erwinia species, Escherichia coli, Hafnia species, Klebsiella species, Morganella species, Proteus vulgaris, Providencia, Salmonella species, Serratia marcescens, and Shigella flexneri), Gardinella family, Haemophilus influenzae, Halobacteriaceae family, Helicobacter family, Legionallaceae family, Listeria species, Methylococcaceae family, mycobacteria (e.g.
  • fungal pathogens include, but are not limited to: Absidia species (e.g., Absidia corymbifera and Absidia ramosa), Aspergillus species, (e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus), Basidiobolus ranarum, Blastomyces dermatitidis, Candida species (e.g., Candida albicans, Candida glabrata, Candida kerr, Candida krusei, Candida par apsilosis, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Candida stellatoidea, and Candida tropicalis), Coccidioides immitis, Conidiobolus species, Cryptococcus neoforms, Cunninghamella species, dermatophytes, Histoplasma capsulatum, Microsporum gypseum, Mucor
  • the disclosure also provides methods of using engineered antibody conjugates to deplete a cell population.
  • methods of the disclosure are useful in the depletion of the following cell types: eosinophil, basophil, neutrophil, T cell, B cell, mast cell, monocytes, endothelial cell and tumor cell.
  • antibodies of the disclosure deplete a respective cell population by at least 5 percent, 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, 50 percent, 55 percent, 60 percent, 65 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, or more as compared to a control non- engineered antibody or a conjugate thereof.
  • the engineered antibodies of the disclosure and conjugates thereof may also be useful in the diagnosis and detection of diseases of symptoms thereof.
  • the compositions of the disclosure may be useful in the monitoring of disease progression.
  • the compositions of the disclosure may be useful in the monitoring of treatment regimens.
  • the compositions of the disclosure are useful for diagnosis in an ex vivo application, such as a diagnostic kit.
  • compositions of the disclosure may be useful in the visualization of target antigens.
  • the target antigens are cell surface receptors that internalize.
  • the target antigen is an intracellular antigen.
  • the target is an intranuclear antigen.
  • the engineered antibodies or antibody-drug conjugates of the disclosure once bound, internalize into cells wherein internalization is at least about 10 percent, at least about 20 percent, at least about 30 percent, at least about 40 percent, at least about 50 percent, at least about 60 percent, at least about 70 percent, at least about 80 percent, or at least about 90 percent, at least about 100 percent, at least about 110 percent, at least about 130 percent, at least about 140 percent, at least about 150 percent, at least about 160 percent, or at least about 170 percent more than control antibodies as described herein.
  • engineered antibody conjugates of the disclosure and the engineered Fc polypeptides, engineered C ⁇ polypeptides, and engineered C ⁇ polypeptides for the treatment of other cancers or autoimmune disorders is also contemplated and within the scope of the present disclosure.
  • the present disclosure provides a composition, for example, but not limited to, a pharmaceutical composition, containing an engineered antibody or engineered antibody conjugate, an engineered Fc polypeptide or conjugate thereof, an engineered Fc fusion protein comprising an engineered Fc region or a conjugate thereof, an engineered C ⁇ polypeptide or a conjugate thereof, and engineered C ⁇ polypeptide or a conjugate thereof, formulated together with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition containing an engineered antibody or engineered antibody conjugate, an engineered Fc polypeptide or conjugate thereof, an engineered Fc fusion protein comprising an engineered Fc region or a conjugate thereof, an engineered C ⁇ polypeptide or a conjugate thereof, and engineered C ⁇ polypeptide or a conjugate thereof, formulated together with a pharmaceutically acceptable carrier.
  • composition is a pharmaceutical composition comprising one or a combination of engineered antibodies, or engineered antibody conjugates of the present disclosure, formulated together with a pharmaceutically acceptable carrier.
  • compositions may include one or a combination of, for example, but not limited to two or more different engineered antibodies of the disclosure.
  • a pharmaceutical composition of the disclosure may comprise a combination of engineered antibodies that bind to different epitopes on the target antigen or that have complementary activities.
  • compositions of the disclosure also can be administered in combination therapy, such as, combined with other agents.
  • the combination therapy can include an engineered antibody or conjugate thereof of the present disclosure combined with at least one other therapy wherein the therapy may be surgery, immunotherapy, chemotherapy, radiation treatment, or drug therapy.
  • the pharmaceutical compounds of the disclosure may include one or more pharmaceutically acceptable salts.
  • Such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a pharmaceutical composition of the disclosure also may include a pharmaceutically acceptable anti-oxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents,
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier.
  • Such drops may further comprise buffering agents, salts, or one or more other of the additional ingredients described herein.
  • Other ophthalmalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form or in a liposomal preparation.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • Other “additional ingredients” which may be included in the pharmaceutical compositions of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, PA (1985 .
  • the engineered antibody or engineered antibody conjugate is administered in an intravenous formulation as a sterile aqueous solution containing 5 mg/m, or more preferably, about 10 mg/ml, or yet more preferably, about 15 mg/ml, or even more preferably, about 20 mg/ml of antibody, with sodium acetate, polysorbate 80, and sodium chloride at a pH ranging from about 5 to 6.
  • the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg/ml of antibody, with 20 mM sodium acetate, 0.2 mg/ml polysorbate 80, and 140 mM sodium chloride at pH 5.5.
  • a solution comprising an engineered antibody or engineered antibody conjugate can comprise, among many other compounds, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene) glycol, EDTA, methionine, and any combination thereof, and many other compounds known in the relevant art.
  • part of the dose is administered by an intraveneous bolus and the rest by infusion of the engineered antibody or engineered antibody conjugate formulation.
  • a 0.01 mg/kg intravenous injection of the engineered antibody or engineered antibody conjugate may be given as a bolus, and the rest of a predetermined engineered antibody or engineered antibody conjugate dose may be administered by intravenous injection.
  • a predetermined dose of the engineered antibody may be administered, for example, over a period of an hour and a half to two hours to five hours.
  • a therapeutic agent where the agent is, e.g., a small molecule, it can be present in a pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions of the disclosure are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever- inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions.
  • endotoxin units EU
  • pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
  • endotoxin and pyrogen levels in the composition are less then about 10 EU/mg, or less then about 5 EU/mg, or less then about 1 EU/mg, or less then about 0.1 EU/mg, or less then about 0.01 EU/mg, or less then about 0.001 EU/mg.
  • the disclosure comprises administering a composition wherein said administration is oral, parenteral, intramuscular, intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal, intravenous, cutaneous, subcutaneous or transdermal.
  • the disclosure further comprises administering a composition in combination with other therapies, such as surgery, chemotherapy, hormonal therapy, biological therapy, immunotherapy or radiation therapy.
  • compositions including an engineered antibody or engineered antibody conjugate of the disclosure are mixed with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y .; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.
  • an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix.
  • an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects.
  • the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub.
  • Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.
  • Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • compositions comprising engineered antibodies or engineered antibody conjugates of the disclosure can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week.
  • Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation.
  • a specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects.
  • a total weekly dose may be at least 0.05 micro g/kg body weight, at least 0.2 micro g/kg, at least 0.5 micro g/kg, at least 1 micro g/kg, at least 10 micro g/kg, at least 100 micro g/kg, at least 0.2 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or at least 50 mg/kg (see, e.g., Yang, et al., 2003, New Engl. J. Med. 349:427-434 ; Herold, et al., 2002, New Engl. J. Med. 346:1692-1698 ; Liu, et al., 1999, J. Neurol.
  • the dose may be at least 15 micro g, at least 20 micro g, at least 25 micro g, at least 30 micro g, at least 35 micro g, at least 40 micro g, at least 45 micro g, at least 50 micro g, at least 55 micro g, at least 60 micro g, at least 65 micro g, at least 70 micro g, at least 75 micro g, at least 80 micro g, at least 85 micro g, at least 90 micro g, at least 95 micro g, or at least 100 micro g.
  • the doses administered to a subject may number at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.
  • the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/'kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight.
  • the dosage of the engineered antibodies or engineered antibody conjugates of the disclosure may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.
  • the dosage of the antibodies of the disclosure may be 150 micro g/kg or less, 125 micro g/kg or less, 100 micro g/kg or less, 95 micro g/kg or less, 90 micro g/kg or less, 85 micro g/kg or less, 80 micro g/kg or less, 75 micro g/kg or less, 70 micro g/kg or less, 65 micro g/kg or less, 60 micro g/kg or less, 55 micro g/kg or less, 50 micro g/kg or less, 45 micro g/kg or less, 40 micro g/kg or less, 35 micro g/kg or less, 30 micro g/kg or less, 25 micro g/kg or less, 20 micro g/kg or less, 15 micro g/kg or less, 10 micro g/kg or less, 5 micro g/kg or
  • Unit dose of the engineered antibodies or engineered antibody conjugates of the disclosure may be 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 m g, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
  • the dosage of the engineered antibodies or engineered antibody conjugates of the disclosure may achieve a serum titer of at least 0.1 micro g/ml, at least 0.5 micro g/ml, at least 1 micro g/ml, at least 2 micro g/ml, at least 5 micro g/ml, at least 6 micro g/ml, at least 10 micro g/ml, at least 15 micro g/ml, at least 20 micro g/ml, at least 25 micro g/ml, at least 50 micro g/ml, at least 100 micro g/ml, at least 125 micro g/ml, at least 150 micro g/ml, at least 175 micro g/ml, at least 200 micro g/ml, at least 225 micro g/ml, at least 250 micro g/ml, at least 275 micro g/ml, at least 300 micro g/ml, at least 325 micro g/ml, at least 350 micro g/ml, at
  • the dosage of the antibodies of the disclosure may achieve a serum titer of at least 0.1 micro g/ml, at least 0.5 micro g/ml, at least 1 micro g/ml, at least, 2 micro g/ml, at least 5 micro g/ml, at least 6 micro g/ml, at least 10 micro g/ml, at least 15 micro g/ml, at least 20 micro g/ml, at least 25 micro g/ml, at least 50 micro g/ml, at least 100 micro g/ml, at least 125 micro g/ml, at least 150 micro g/ml, at least 175 micro g/ml, at least 200 micro g/ml, at least 225 micro g/ml, at least 250 micro g/ml, at least 275 micro g/ml, at least 300 micro g/ml, at least 325 micro g/ml, at least 350 micro g/ml, at least 375 micro
  • Doses of engineered antibodies or engineered antibody conjugates of the disclosure may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.
  • An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects (see, e.g., Maynard, et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, F la.; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK ).
  • the route of administration may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et al., 1983, Biopolymers 22:547-556 ; Langer, et al., 1981, J. Biomed. Mater. Res. 15: 167-277 ; Langer, 1982, Chem. Tech. 12:98-105 ; Epstein, et al., 1985, Proc. Natl. Acad. Sci.
  • composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
  • an engineered antibody or engineered antibody conjugate, combination therapy, or a composition of the disclosure is administered using Alkermes AIRTM pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).
  • a composition of the present disclosure may also be administered via one or more routes of administration using one or more of a variety of methods known in the art.
  • routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • a composition of the disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20 ; Buchwald et al., 1980, Surgery 88:501 ; Saudek et al., 1989, N. Engl. J. Med. 321:514 ).
  • Polymeric materials can be used to achieve controlled or sustained release of the therapies of the disclosure (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974 ); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984 ); Ranger and Peppas, 1983, J., Macromol. ScL Rev. Macromol. Chem. 23:61 ; see also Levy et al, 1985, Science 11 225:190 ; During et al., 19Z9, Ann. Neurol. 25:351 ; Howard et al, 1989, J. Neurosurg.
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co- vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N -vinyl pyrrolidone), polyvinyl alcohol), polyacrylamide, polyethylene glycol), polylactides (PLA), polyoeactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984 )).
  • Controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533 . Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies of the disclosure or conjugates thereof. See, e.g., U.S. Pat. No. 4,526,938 , International Patent Publication Nos.
  • the engineered antibody or engineered antibody conjugate of the disclosure can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995 ).
  • viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity, in some instances, greater than water are typically employed.
  • Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which are, if desired, sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, such as, for example, osmotic pressure.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts
  • Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle.
  • a pressurized volatile e.g., a gaseous propellant, such as freon
  • humectants can also be added to
  • compositions comprising engineered antibodies or engineered antibody conjugates are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops.
  • prophylactic or therapeutic agents for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • a second therapeutic agent e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation
  • a second therapeutic agent e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation
  • An effective amount of therapeutic may decrease the symptoms by at least 10 percent; by at least 20 percent; at least about 30 percent; at least 40 percent, or at least 50 percent.
  • Additional therapies which can be administered in combination with the engineered antibodies or engineered antibody conjugates of the disclosure, may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the antibodies of the disclosure.
  • the engineered antibodies or engineered antibody conjugates of the disclosure and the other therapies may be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally, followed by the administration of a third therapy (e.g., prophylactic or therapeutic agent) for a period of time and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and/or to improve the efficacy of the therapies.
  • a first therapy e.g., a first prophylactic or therapeutic agent
  • a second therapy e.g., a second prophylactic or therapeutic agent
  • a third therapy e.g., prophylactic or therapeutic agent
  • the engineered antibodies or engineered antibody conjugates of the disclosure can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the disclosure cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811 ; 5,374,548 ; and 5,399,331 .
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade, 1989, J. Clin. Pharmacol.
  • Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 ); mannosides ( Umezawa et al., Biochem. Biophys. Res. Commun. 153: 1038 ); antibodies ( P. G. Bloeman et al., 1995, FEBS Lett. 357: 140 ; M. Owais et al., 1995, Antimicrob. Agents Chemother. 39: 180 ); surfactant protein A receptor ( Briscoe et al. (1995) Am. J. Physiol. 1233: 134 ); pl20 ( Schreier et al. (1994) J. Biol. Chem. 269:9090 ); see also K. Keinanen; M.L. Laukkanen, 1994, FEBS Lett. 346:123 ; Killion; Fidler, 1994; Immunomethods 4:273 .
  • the disclosure provides protocols for the administration of pharmaceutical composition comprising engineered antibodies or engineered antibody conjugates of the disclosure alone or in combination with other therapies to a subject in need thereof.
  • the therapies e.g., prophylactic or therapeutic agents
  • the therapy e.g., prophylactic or therapeutic agents
  • the combination therapies of the present disclosure can also be cyclically administered.
  • Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g., agents), and/or to improve, the efficacy of the therapies.
  • a first therapy e.g., a first prophylactic or therapeutic agent
  • a second therapy e.g., a second prophylactic or therapeutic agent
  • the therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the disclosure can be administered to a subject concurrently.
  • the term "concurrently” is not limited to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather it is meant that a pharmaceutical composition comprising engineered antibodies or engineered antibody conjugates of the disclosure are administered to a subject in a sequence and within a time interval such that the antibodies of the disclosure or conjugates thereof can act together with the other therapy(ies) to provide an increased benefit than if they were administered otherwise.
  • each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.
  • Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route.
  • the therapies are administered to a subject less than 15 minutes, less than 30 minutes, less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart.
  • two or more therapies are administered to a within the same patient visit.
  • the prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition.
  • the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions.
  • the prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.
  • ADCs antibody drug conjugates
  • DAR molar drug:antibody ratio
  • the data disclosed herein demonstrate an approach whereby reactive cysteine residues were engineered into the antibody constant regions (e.g., heavy and light chain constant regions) to facilitate generation of homogeneous ADCs with drug:antibody ratio of either 2 or 4 and the successful use of these novel antibodies as a useful platform for site-specific conjugation for various therapeutic targeting moieties.
  • antibody constant regions e.g., heavy and light chain constant regions
  • the amino acid sequence of wild type IgG1 without mutations and with numbering in sequential order (starting at alanine 1 and ending in lysine 330) is as follows, and is designated as SEQ ID NO:1:
  • Table 2 sets forth the location of the mutations relative to wild type endogenous human IgG1 wherein the amino acid residue was mutated to cysteine for thiol reactive site-specific conjugation. Table 2 indicates the positions where human IgG1 residues were replaced with reactive cysteines. Positions are identified using the EU index numbering system as set forth in Kabat et al. (1991, NIH Publication 91 - 3242, National Technical Information Service, Springfield, VA) and also according to sequential numbering relative to the sequence of SEQ ID NO:1.
  • Single reactive cysteine residues were introduced into a humanized antibody comprising humanized heavy and light chain variable domains specifically binding human 5T4 and a human IgG1 Fc-region (the antibody referred to herein as anti-5T4 or simply "5T4").
  • the reactive cysteine residues were introduced into the IgG1 Fc at the twelve positions listed in Table 2 using an over-lapping PCR mutagenesis method.
  • the amino acid sequence of wild type human IgG1, without mutations, is set forth in SEQ ID NO:1.
  • PCR mutagenesis was performed as follows. Sense and anti-sense mutagenic oligonucleotides harboring the individual cysteine mutations as well as forward and reverse human IgG1 constant region flanking primers were synthesized at Integrated DNA Technologies, Inc (ParkCoralville, Iowa).
  • PCR reaction 1 contained one hundred nanograms (ng) of anti-5T4 antibody encoding plasmid DNA, 100 pmoles forward flanking primer oligonucleotide, 100 pmoles anti-sense mutagenic oligonucleotide, 1 ⁇ l Vent® polymerase (New England Biolabs Inc., Ipswich, Massachusetts), 25 ⁇ l 2x HN PCR buffer (EPICENTRE® Biotechnologies, Madison, WI) and H 2 O to bring the volume of the reaction to 50 ⁇ l.
  • ng nanograms
  • PCR reaction 2 was made by mixing 100 ng A1 anti-5T4 antibody encoding plasmid DNA, 100 pmoles sense mutagenic oligonucleotide, 100 pmoles reverse flanking primer oligonucleotide, 1 ⁇ l Vent® polymerase, 25 ⁇ l 2x HN PCR buffer and adding H 2 O to bring the volume of the reaction to 50 ⁇ l.
  • the PCR parameters for reactions 1 and 2 were 95°C for 1 minute, 63°C for 1 minute, 72°C for 1 minute for 25 cycles and then 10 minutes at 72°C.
  • the final PCR reaction was done by mixing 1 ⁇ l each of PCR reactions 1 and 2, 100 pmoles each of the forward and reverse flanking primer oligonucleotides, 1 ⁇ l Vent® polymerase, 25 ⁇ l 2x HN PCR buffer and H 2 O to bring the volume of the reaction to 50 ⁇ l.
  • the final PCR reaction parameters were the same as used for reactions 1 and 2.
  • the human IgG1 variants harboring the individual engineered cysteine residues were joined to the A1 heavy chain variable region using T4 DNA Ligase (New England Biolabs Inc., Ipswich, Massachusetts) and the nucleic acid was sequence confirmed.
  • COS-1 cells were transiently co-transfected with plasmid DNA encoding the cysteine variants and the parental anti-5T4 antibody, i.e., the wild type IgG1 Fc region that did not comprise any mutations, using standard methods. After a period of 48 hours, the cell culture medium was harvested and the resultant conditioned medium containing the 5T4-cysteine antibody variants was quantitated by total human IgG sandwich ELISA.
  • a flat bottom ELISA plate (Costar catalog #3590) was coated overnight at room temperature with 100 ⁇ l each well of 1 ⁇ /ml goat anti-human IgG in PBS (Thermo/Pierce catalog #31125). Plates were blocked with 100 ⁇ l/well of a 0.02% Casein Solution in PBS for a minimum of 3 hours or up to 24 hours at room temperature. Standards and samples were serially diluted in assay buffer (0.5% BSA+0.02%Tween-20 in PBS) and 100 ⁇ l was added to the coated/blocked ELISA plate and incubated for 3 to 24 hours at room temperature.
  • CHO cells were co-transfected with heavy and light chain DNA encoding eight (S254C, T359C, E380C, K392C, L398C, V422C, S440C and L443C) anti-5T4 antibody single cysteine variants and stable high production pools were isolated using standard procedures well-known in the art. DNA was co-transfected into the CHO cells since the heavy and light chain expression constructs were on separate expression plasmids. The CHO pool for the parental anti-5T4 antibody was also generated by co-transfecting heavy and light chain expression constructs into CHO cells.
  • a standard two step purification strategy i.e., Protein-A affinity capture followed by size exclusion chromatography (SEC) was used to isolate these cysteine variants from the concentrated CHO pool starting material.
  • SEC size exclusion chromatography
  • the ability to isolate the antibodies using this two-step process demonstrated that the Fc region Protein A binding site was not altered by the presence of the engineered cysteine and that the mutated IgG1 Fc region bound Protein A similarly to wild type IgG1 Fc.
  • Minimal high molecular weight aggregated species were detected following elution from Protein A resin for 6 of the 8 single-cysteine variants and this species is reported as percent peak of interest (%POI) in Table 4.
  • 5T4 binding properties were assessed for the anti-5T4 antibody variants comprising single-cysteine variants using a competition ELISA assay with biotinylated wild type anti-5T4 antibody comprising the wild-type human IgG1 constant region as the reporter antibody to determine if the 5T4 cysteine variants could effectively compete with this wild type 5T4 antibody for binding to 5T4 antigen.
  • the parental anti-5T4 reporter antibody (comprising wild type IgG1 Fc without mutations) was biotinylated using EZ-link Sulfo-NHS-Biotin Sulfosuccinimidobiotin (Thermo/Pierce, catalog number 21217) at a molar coupling ratio of 20:1 according to the manufacturer's protocols.
  • Protein for this assay was generated by transiently transfecting DNA encoding the anti-5T4 single-cysteine variants and the wild type anti-5T4 antibody into COS-1 cells. That is, both the variants and the control wild type antibodies comprised human IgG1 Fc regions.
  • the resultant conditioned medium containing the anti-5T4 single-cysteine variants and the anti-5T4 wild type gG1 antibody was assayed using a total human IgG sandwich ELISA as previously described.
  • a 96-well plate (Costar catalog #3590) was coated with human truncated recombinant 5T4 protein (5T4-tm - _myc_his) lacking the transmembrane and intracellular domains of 5T4 (see Boghaert et al., 2008, Int. J. Oncol. 32:221-234 ) and further comprising Myc and histidine tags.
  • the 5T4-tm - _myc_his construct was diluted to 1 ⁇ g/ml in PBS-CMF pH 7.2, 100 ⁇ l was added to each well of the plate, and the plate was incubated overnight at 4°C. The contents of the plate were discarded and then the plate was blocked with PBS-CMF pH7.2 + 0.02% casein for 3 hours at room temperature.
  • Biotinylated anti-5T4 antibody at 20 ng/ml in PBS + 0.5% BSA + 0.02% tween-20 was mixed with varying concentrations of the anti-5T4 single-cysteine variants or wild type anti-5T4 antibody as the positive control,the samples were added to the 5T4 coated-blocked plate and incubated at room temperature for 2 hours.
  • each of the antibodies used in this assay comprise the identical humanized anti-5T4 V L and V H domains, but the biotinylated reporter antibody comprises a wild type IgG Fc region without an engineered cysteine while the competitor antibodies comprise either the wild type IgG1 Fc region or mutated IgG1 Fc regions comprising a single-cysteine mutation.
  • antibodies contain inter and intra-chain disulfide bonds that link the four peptide chains and all of these canonical disulfide bonds should be formed for proper antibody folding.
  • the presence of free sulfhydryl (-SH) groups may result in a molecule that is partially unfolded or improperly folded, leading to a heterogeneous population of antibody and decreased protein stability.
  • the fluorescent reagent ThioGlo®1 EMD Millipore, a dye that binds using maleimide chemistry, was used to detect free sulfhydryl groups for the anti-5T4 single-cysteine variants.
  • N-acetyl-L-cysteine was used as a standard to quantify the amount of free sulfhydryl present for each antibody.
  • Bovine serum albumin (BSA; OmniPur BSA Fraction V, Catalog No. 2910, EMD Chemicals) was additionally included as a positive control since it contains one single unpaired cysteine.
  • BSA OmniPur BSA Fraction V, Catalog No. 2910, EMD Chemicals
  • Table 5 the results disclosed herein indicate that free sulfhydryl was detected for the anti-5T4 single-cysteine variants and not for the parental anti-5T4 antibody. In the absence of DTT, increased levels of free -SH was not observed for the six cysteine variants assessed compared with the wild type anti-5T4 protein.
  • FcRn interacts with IgG regardless of subtype in a pH dependent manner and protects the antibody from degradation by preventing it from entering the lysosomal compartment where it is degraded. Therefore, a consideration for selecting positions for introduction of reactive cysteines into the wild type IgG1-Fc region was to avoid altering the FcRn binding properties and half-life of the antibody comprising the engineered cysteine.
  • BIAcore® analysis was performed to determine the steady-state affinity (KD) for anti-5T4 engineered single cysteine variants for binding human FcRn.
  • KD steady-state affinity
  • BIAcore® technology utilizes changes in the refractive index at the surface layer of a sensor upon binding of the anti-5T4 single engineered cysteine variants to human FcRn protein immobilized on the layer. Binding is detected by surface plasmon resonance (SPR) of laser light refracting from the surface.
  • SPR surface plasmon resonance
  • Human FcRn was specifically biotinylated through an engineered Avi-tag using the BirA reagent (Catalog #: BIRA500, Avidity, LLC, Aurora, Colorado) and immobilized onto a streptavidin (SA) sensor chip to enable uniform orientation of the FcRn protein on the sensor.
  • SA streptavidin
  • various concentrations of the anti-5T4 single-cysteine variants in 20mM MES (2-(N-morpholino)ethanesulfonic acid pH 6.0, with 150 mM NaCl, 3 mM EDTA (ethylenediaminetetraacetic acid), 0.5% Surfactant P20 (MES-EP) were injected over the chip surface.
  • the surface was regenerated using HBS-EP + 0.05% Surfactant P20 (GE Healthcare, Piscataway, NJ, Piscataway, NJ), pH 7.4, between injection cycles.
  • the steady-state binding affinities were determined for the anti-5T4 engineered single-cysteine variants and these were compared with the parental wild type anti-5T4 (comprising no cysteine mutations in the IgG1 Fc region).
  • the results are set forth at Table 6, and these data demonstrate that incorporation of engineered cysteine residues into the IgG-Fc region at the novel positions tested did not alter affinity to FcRn. TABLE 6.
  • ADCs were prepared by conjugating mcMMAD, as more fully disclosed below, to the engineered cysteine for the variants 5T4-E380C, 5T4-L398C, 5T4-V422C and 5T4-L443C.
  • COS-1 cells were transiently co-transfected with heavy and light chain DNA encoding the 5T4 double cysteine variants and the parental 5T4 antibody. After a period of 48 hours, each cell culture medium was assayed to determine the level of human IgG1 antibody expressed for each construct using the total human IgG sandwich ELISA described previously. As shown in Table 9, each double cysteine engineered anti-5T4 antibody variant expressed at a comparable level compared with the parental anti-5T4 antibody not comprising any additional cysteines.
  • 5T4 antibody Amount of human IgG1 in the cell culture medium [ ⁇ g/ml] Wild type 5T4 41.2 5T4-E380C+L443C 32.6 5T4-E380C+L398C 45.4 5T4-L398C+ L443C 52.0 5T4-E380C+V422C 39.6 5T4-V422C+L443C 42.2 5T4-L398C+V422C 44.0
  • HEK-293 cells were transiently co-transfected with heavy and light chain DNA encoding the six 5T4 double-cysteine engineered antibody variants using standard methods.
  • the double-cysteine variant antibodies were purified using a standard two step purification strategy, Protein-A affinity capture followed by size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • nucleic acid encoding the anti-Her2 antibody human IgG1 constant region was removed from the vector by restriction enzyme digestion and replaced with a nucleic acid encoding human heavy chain constant IgG1 Fc regions comprising the single and double engineered cysteine residues using T4 DNA Ligase (New England Biolabs Inc., Ipswich, Massachusetts). The resulting nucleic acid was sequence confirmed for each construct.
  • Antibodies were successfully produced for use in conjugation studies by transiently co-transfecting COS-1 cells with heavy and light chain DNA encoding the anti-Her2 single and double cysteine engineered antibody variants demonstrating that the antibodies could be transiently expressed in cells. Further, the antibodies were purified using a standard two step purification strategy, Protein-A affinity capture followed by size exclusion chromatography (SEC). The data disclosed herein (Table 12) demonstrate that low levels of high molecular weight (HMW) aggregated species were detected following elution from Protein A resin for all 6 anti-Her2 double cysteine variants and that this HMW species could be removed using size exclusion chromatography.
  • HMW high molecular weight
  • nucleic acid sequence encoding the wild type human IgG1 Fc constant region of an anti-human VEGFR2 antibody was removed by restriction enzyme digestion and replaced with a nucleic acid sequence encoding the heavy chain constant region comprising a single cysteine residue at position L443C (SEQ ID NO:72) using T4 DNA Ligase (New England Biolabs Inc., Ipswich, Massachusetts) and the resulting nucleic acid was sequence confirmed.
  • the antibody is produced by transfecting COS-1 with the nucleic acid encoding the antibody and the protein is purified using a two-step (Protein A followed by SEC chromatographies) process. This demonstrates that the anti-human VEGFR2 antibody comprising the single reactive cysteine can be expressed and that the Protein A binding of the Fc region is not affected.
  • cysteine residues with either an optimal predicted pKa range between 9.5 and 11.5, and/or a predicted side chain solvent accessibility between 15 and 60 may mimic the properties of the conjugated cysteine mutants disclosed previously herein, including, but not limited to E380C, K392C, L398C, V422C and L443C. Since these properties (pKa and predicted side chain solvent accessibility) are correlated, it is difficult to establish which criteria are associated with the desired biological outcomes, including, but not limited to, low propensity to aggregate and facile conjugation to linkers and payloads.
  • novel ADCs disclosed previously demonstrating successful conjugation of the antibodies comprising novel IgG1 Fc regions comprising engineered reactive cysteines were prepared as now described herein below.
  • the resulting material was diluted to 1 mL in PBS containing 50 mM EDTA and treated with 16 uL of a 5 mM solution of mcMMAD in dimethyl acetamide (DMA) (approximately 5 equivalents). After standing at room temperature (about 23 degrees C) for 1.5 hour the reaction tube was centrifuged to concentrate the material to approximately 100 ⁇ L. The mixture was diluted to 1 mL with PBS. This process was repeated 2 times in order to remove the excess maleimide reactant (e.g., mcMMAD).
  • DMA dimethyl acetamide
  • the antibody conjugates were generally purified and characterized using size-exclusion chromatography (SEC) as described below.
  • SEC size-exclusion chromatography
  • the loading of the drug onto the intended site of conjugation was determined using a variety of methods including mass spectrometry (MS), reverse phase HPLC, and hydrophobic interaction chromatography (HIC), as more fully described below.
  • MS mass spectrometry
  • HPLC reverse phase HPLC
  • HIC hydrophobic interaction chromatography
  • Antibody-drug conjugates (Ab-linker-payload, e.g., Ab-mcMMAD and Ab-vcMMAD) were generally purified using SEC chromatography using a Waters Superdex200 10/300GL column on an Akta Explorer FPLC system in order to remove protein aggregate and to remove traces of payload-linker left in the reaction mixture.
  • ADCs were free of aggregate and small molecule prior to SEC purification and were therefore not subjected to preparative SEC.
  • the eluent used was PBS at 1 mL/min flow. Under these conditions, aggregated material (eluting at about 10 minutes at room temperature) was easily separated from non-aggregated material (eluting at about 15 minutes at room temperature).
  • Hydrophobic payload-linker combinations frequently resulted in a "right-shift" of the SEC peaks.
  • this SEC peak shift may be due to hydrophobic interactions of the linker-payload with the stationary phase.
  • this right-shift allowed for conjugated protein to be partially resolved from non-conjugated protein.
  • Analytical size-exclusion chromatography Analytical SEC was carried out on an Agilent 1100 HPLC using PBS as eluent. The eluent was monitored at 220 and 280 nM. The methods utilized are as follows:
  • Table 15 sets forth the results for various antibody-drug conjugates purified and characterized using the above methods. The loading analysis and MS characterizations are discussed below.
  • the conjugates were analyzed by analytical SEC in order to establish the integrity of the purified protein conjugate and to ensure that minimal aggregation occurred during the conjugation.
  • the two methods described above give approximately equivalent retention times and were used in different circumstances simply for practical purposes such as column availability and reliability. Generally it was observed that aggregated material induced a leftward shift of the retention time of approximately one minute. Examples of two analytical SEC traces are illustrated in Figure 2.
  • Figure 2A shows the SEC tracing for 5T4-L398C-mcMMAD (using method SEC-A);
  • Figure 2B shows the SEC trace for 5T4-V422C-vcMMAD (using method SEC-B). These tracings show that the material is non-aggregated and contains no measurable small molecule contaminant.
  • Samples were prepped for LCMS analysis by combining approximately 20 uL of sample (approximately 1 mg/mL ADC in PBS) with 20 uL of 20 mM dithiothreitol (DTT). After allowing the mixture to stand at room temperature for 5 minutes, the samples were injected into an Agilent 1100 HPLC system fitted with a Agilent Poroshell 300SB-C8 (2.1x75mm) column. The system temperature was set to 60°C. A 5 minute gradient from 20% to 45% acetonitrile in water (with 0.1% formic acid modifier) was utilized.
  • the eluent was monitored by UV (220 nM) and by a Waters MicromassZQ mass spectrometer (ESI ionization; cone voltage: 20V; Source temp: 120°C; Desolvation temp: 350°C).
  • ESI ionization cone voltage: 20V
  • Source temp 120°C
  • Desolvation temp 350°C
  • the crude spectrum containing the multiple-charged species was deconvoluted using MaxEnt1 within MassLynx 4.1 software package according to the manufacturer's instructions.
  • the spectra for the entire elution window (usually 5 minutes) are combined into a single summed spectra (i.e., a mass spectrum that represents the MS of the entire sample).
  • MS results for ADC samples were compared directly to the corresponding MS of the identical non-loaded control antibody. This allows for the identification of loaded/nonloaded heavy chain (HC) peaks and loaded/nonloaded light chain (LC) peaks.
  • the ratio of the various peaks can be used to establish loading based on the equation below (Equation 1). Calculations are based on the assumption that loaded and non-loaded chains ionize equally which has been determined to be a generally valid assumption.
  • Equation 2 is used to estimate the amount of loading onto non-engineered cysteine residues
  • loading onto the light chain (LC) was considered, by definition, to be nonspecific loading.
  • loading only the LC was the result of inadvertent reduction of the HC-LC disulfide bridge (i.e., the antibody was "over-reduced").
  • cysteine mutants disclosed in Tables 14-16 are located in the CH2 and CH3 domains within the Fc domain of the IgG1 heavy chain. Any nonspecific loading of the electrophillic payload onto the antibody is presumed to occur at the "interchain” also referred to as the "internal” cysteine residues (i.e., those that are typically part of the HC-HC or HC-LC disulfide bridges).
  • the conjugates were treated with a protease known to cleave between the Fab domains and the Fc domain of the antibody.
  • a protease known to cleave between the Fab domains and the Fc domain of the antibody.
  • One such protease is the cysteine protease IdeS, marketed as "FabRICATOR®” by Genovis, and described in von Pawel-Rammingen et al., 2002, EMBO J. 21:1607 .
  • Figure 4 depicts a diagram illustrating the cleavage by this protease of an intact antibody molecule showing the positions (dark squares) of the internal cystine bonds.
  • the ADC was treated with FabRICATOR® protease and the sample was incubated at 37°C for 30 minutes.
  • Samples were prepped for LCMS analysis by combining approximately 20 ⁇ L of sample (approximately 1 mg/mL in PBS) with 20 ⁇ L of 20 mM dithiothreitol (DTT) and allowing the mixture to stand at room temperature for 5 minutes.
  • DTT dithiothreitol
  • FIG. 5A shows the MS tracing results for 5T4-L443C variant that is not loaded after FabRICATOR® protease treatment.
  • the insert depicts a diagram illustrating the proteolytic cleavage fragments generated by FabRICATOR® treatment.
  • Figure 5B is a graph showing the MS tracings results for FabRICATOR® treatment of ADC 5T4-L443C-mcMMAD.
  • the insert shows a diagram illustrating the fragments resulting from proteolytic cleavage and illustrating that the linker and payload are associated with the C-terminal HC fragment indicating that loading is located at the reactive cysteine introduced by mutation.
  • the results for the analysis of a subset of the ADCs are set forth in Table 17.
  • Samples were prepped for reverse-phase HPLC analysis by combining approximately 20 uL of sample (approximately 1 mg/mL in PBS) with 20 uL of 20 mM dithiothreitol (DTT). After allowing the mixture to stand at room temperature for 5 minutes, the samples were injected into an Agilent 1100 HPLC system fitted with an Agilent Poroshell 300SB-C8 (2.1x75mm) column. The system temperature was set to 60° C and the eluent was monitored by UV (220 nM and 280 nM).
  • Figure 6 shows reverse phase HPLC traces under reducing conditions for (A) unmodified wild type anti-5T4 antibody; (B) 5T4-E380C-mcMMAD; and (C) 5T4-L443C-mcMMAD.
  • the results obtained with reverse phase HPLC are consistent with those obtained using MS analysis as disclosed previously herein.
  • the specific loading and non-specific loading were calculated for each sample using the AUC for each indicated peak in Figure 6 . The loading values thus obtained are consistent with the previous loading calculations.
  • Figure 7 shows HIC traces produced for several variants illustrating the distribution of variously loaded antibody species.
  • Figure 7 depicts traces for (A) control anti-5T4-L443C non-loaded antibody; (B) 5T4-L443C-vcMMAD; (C) 5T4-E380C-vcMMAD; and (D) 5T4-E380C-mcMMAD.
  • the loaded antibody can easily be baseline separated from nonloaded antibody using the described method.
  • differentially loaded species can typically (but not always) be resolved.
  • the AUC for the various peaks shown in Figure 7 was used to calculate loading values based on HIC and to complement and further verify the loading values determined by other methods previously described.
  • the loadings thus calculated are set forth in Table 18 which compares the loading estimations produced using HIC methodology and MS methodology. As can be seen, there is a very tight correlation between loading values calculated using the two different methods.
  • the above methodology provides several independent methods for establishing the loading of electrophilic payload-linkers onto the engineered Cys residues. These methods are complementary, consistent, and independent of one another. The combination of these methods allows the loading estimates to be determined even in the face of complicating factors such as payloads that may contain functionality that results in unusual MS ionization or high UV absorption. These data demonstrate that the ADCs comprising a reactive cysteine at a novel position in the IgG1 Fc region provide a useful platform for production of potentially therapeutically effective ADCs demonstrating a precise DAR which can be carefully controlled and measured.
  • Method B Reduction/reoxidation method for the conjugation of maleimide payloads to single and double cys-mutants using an alternative conjugation method
  • Method B In order to improve homogeneity of loading, an alternative procedure (“Method B”) involving complete reduction of the engineered antibody with TCEP (tris2-carboxyethyl)phosphine) followed by re-oxidation of the internal disulfides with DHA (dehydroascorbic acid) was used which allowed for a conjugation with maleimides that resulted in a more homogeneous ADC (as measured by MS and by HIC).
  • TCEP tris2-carboxyethyl)phosphine
  • DHA dehydroascorbic acid
  • Figures 8 and 9 show the tracings produced by conjugations using Method “A” ( Figures 8A, 8C, 8E, 8G , 9A, 9C, 9E and 9G ) and conjugations using "Method B” ( Figures 8B, 8D, 8F, 8H , 9B, 9D, 9F, and 9H ).
  • 8 conjugates are as follows: 8A and 8B, 5T4-E380C-mcMMAD; 8C and 8D, 5T4-L398C-mcMMAD; 8E and 8F, 5T4-L443C-mcMMAD; 8G and 8H, 5T4-K388C-mcMMAD; 9A and 9B, 5T4-E380C+L398C-mcMMAD; 9C and 9 D, 5T4-E398C+L443C-mcMMAD; 9E and 9F, 5T4-E380C+L443C-mcMMAD; and 9G and 9H, 5T4-E380C+V422C-mcMMAD.
  • Method B was performed as follows. A 20 mM TCEP solution (50 to 100 molar equivalents) was added to the antibody (5 mg) such that the final antibody concentration was 5 mg/mL in PBS containing 50 mM EDTA. After allowing the reaction to stand at 37° C for 1.5 hour, the antibody was buffer exchanged into PBS containing 50 mM EDTA using a 50 kD MW cutoff spin concentration device (3x3 mL wash, 10x concentration per cycle).
  • the antibody/DHA mixture was buffer exchanged into PBS containing 50 mM EDTA using a 50 kD MW cutoff spin concentration device (3x3mL wash, 10x concentration per cycle).
  • the resulting antibody was re-suspended in 1 mL of PBS containing 50 mM EDTA and treated with 33 uL of 10 mM maleimide payload (mcMMAD) in DMA.
  • mcMMAD 10 mM maleimide payload
  • the material was buffer exchanged (as above) into 1 mL of PBS (3x3 mL washes, 10x concentration per cycle). Purification by SEC was performed (as needed) to remove any aggregated material.
  • MDAMB435/5T4 transfected cells expressing human 5T4 antigen and control MDAMB435/neo cell that were not transfected were prepared as described previously ( Boghaert et al., 2008, Int. J. Oncol. 32:221-234 ).
  • Raji (CCL-86) cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines were determined to be mycoplasma free as determined by a polymerase chain reaction mycoplasma detection assay (ATCC, Manassas, VA).
  • the cell line MDAMB453/5T4 was maintained in MEM medium with Earl's salts supplemented with 10% fetal bovine serum (FBS), 1% MEM non essential amino acids and 1% MEM vitamins, 1 mM sodium pyruvate, penicillin G sodium 100 U/ml, streptomycin sulfate 100 ⁇ g/ml and 2 mM L-Glutamine plus 1.5mg/mL of selection antibiotic G418.
  • Raji cell line was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 1 mM sodium pyruvate, 0.2% glucose, penicillin G sodium 100 U/ml, streptomycin sulfate 100 ⁇ g/ml and 2 mM L-Glutamine. Before using Raji, viable cells were isolated by density-gradient centrifugation (30 min at 1000xg) using Lymphoprep (Nycomed, Oslo, Norway).
  • mice Female nu/nu (nude) mice (18-23 g) were obtained from Charles River Laboratories, Wilmington, MA. All procedures using mice were approved by the Wyeth Animal Care and Use Committee according to established guidelines.
  • Cells expressing 5T4, and the negative control Raji cells were plated at a density of 500,000 cells/well on non-tissue culture treated 96 well plates and kept on ice. Dilutions of the primary antibody were made in 3% BSA in dPBS (Dulbecco's phosphate buffered saline, 100 mM phosphate, pH 7.4) and added to the plate at a final concentration of 10 ⁇ g/mL. The plates were then incubated on ice for 1 hour followed by 2 washes with 1X DPBS. The secondary antibody, PE conjugated Goat Anti-Human IgG Fc (Jackson ImmunoResearch Labs #109-115-098), was added to the wells at 1:100 dilution.
  • MDAMB435/5T4 cells were plated at 10,000 cells in black 96 well plates.
  • the primary antibody was added at a final concentration of 1 ⁇ g/mL.
  • the plates were then incubated on ice for 1 hour, washed twice with 1X DPBS and then incubated at 4°C in cold media for another hour (this is referred to herein as the "binding plate").
  • binding plate For internalization studies, internalization plates were incubated at 37°C for either 1, 4, or 20 hours. The plates were washed once with 1X DPBS.
  • the secondary antibody peroxidase-conjugated Affinity Pure Goat Anti-Human IgG Fc (Jackson ImmunoResearch Labs #109-035-008), was added to the wells at 1:4000 dilution. After one hour incubation at 4°C with the secondary antibody, the plates were washed thrice and the substrate, LumiGLO® (Cat. No. #54-61-01, Kirkegaard & Perry Labs., Gaithersburg, MD) was added. The difference in average relative fluorescence between the binding plate and the internalization plate was expressed as percentage of binding to estimate the internalization of the antibody.
  • the effect of the ADCs on cell lines was assessed using a cellular viability indicator assay, CellTiter 96® AQ ueous Non-Radioactive Cell Proliferation Assay (MTS) (Promega, Madison, WI), to determine the number of surviving cells following exposure to various ADC treatments.
  • Cells were seeded in 96-well microtiter plates at a density of 5,000 to 10,000 cells per well and exposed to various concentrations of antibody or ADC.
  • the IC 50 of each treatment was calculated based on the logistic regression parameters derived from the dose-response curves. IC 50 s were calculated by logistic non-linear regression and are reported as the concentration (nM) from each treatment group that causes 50% loss of cell viability.
  • ADCC Antibody dependent cellular cytotoxicity
  • PBMC peripheral blood mononucleocytes
  • assay buffer RPMI 1640 supplemented with 10 mM HEPES
  • Target cells MDAMB435/5T4 or MDAMB435/neo control cells were seeded at a density of 1 x 10 4 cells/well in a 96 well assay plate.
  • Antibody or ADCs were added, then human PBMC effector cells (5 x 10 5 ) were dispensed into the wells for an effector:target cell ratio (E:T) of 50:1.
  • the assay plate was incubated at 37 °C for 4 hours for ADCC activity.
  • the plate was harvested by adding equal volume of CytoTox-One reagent (Promega). Stop solution (Promega; 50 ⁇ l) was added to each well and lactate dehydrogenase release was quantified by measuring fluorescence intensity.
  • effector spontaneous corresponds to the signal measured in the presence of PBMC alone
  • target spontaneous corresponds to the signal measured in the presence of target cells alone
  • target maximum corresponds to the signal measured in the presence of detergent-lysed target cells alone.
  • Binding to 5T4 on cells by non-conjugated anti-5T4 variants is equivalent to binding by non-conjugated wild type anti-5T4 parental antibody:
  • Binding of the anti-5T4 IgG1 single-cysteine mutant antibodies (L443C, E380C, L398C, V422C, T359C, S254C, S440C, and K392C), all non-conjugated "naked” antibodies, to 5T4 expressed on the membrane of 5T4 + cell line MDAMB435/5T4 was demonstrated as shown in Figure 11 .
  • Binding of each of the non-conjugated cys mutant 5T4 Abs was similar to the wild type non-conjugated 5T4 IgG1 Ab (labeled as "wtlgG1") at both concentrations tested (1 ⁇ g/ml and 10 ⁇ g/ml).
  • Antibody drug conjugates were prepared using four (4) of the 5T4 cysteine mutants: E380C, L398C, L443C, and V422C.
  • the ADC was prepared by conjugating the mutated antibodies to mcMMAD and vcMMAD as previously disclosed herein (see Example 7).
  • Figure 12A depicts a graph demonstrating that for 5T4-L398C-mcMMAD, 5T4-443C-mcMMAD and 5T4-V422C-mcMMAD, binding of the ADC to the 5T4-positive cell lines , on average for the three (3) ADC concentrations tested (1, 3 and 10 ⁇ g/kg), was similar to that of the native, unconjugated 5T4 antibody.
  • Another critical property for an ADC activity is to be rapidly internalized whilst conjugated to a toxin in order to deliver the toxic payload to the intracellular lysosomal compartment.
  • the novel cysteine mutant ADCs of the present disclosure were subjected to more rigorous and appropriate tests to determine whether they would be internalized whilst conjugated to a true representative linker and cytotoxic payload combination (e.g., mcMMAD) with comparable efficiency when compared to internalization of the parental antibody comprising wild type-IgG1 conventionally conjugated to the same linker-payload combination.
  • the results disclosed herein demonstrate that the novel Cys mutant ADCs were internalized with comparable efficiency to the parental control ADC.
  • non-conjugated 5T4-IgG1 parental antibody or Cys mutant ADC (E380C, L398C, L443C, and the IgG1 parental antibody, each conjugated to mcMMAD) was incubated with MDAMB435/5T4 cells at 37°C for 4 hours at a concentration of 2.25 ⁇ g/ml, the ADC was modulated (i.e., internalized in that it was no longer detected at the cell membrane) in a time-dependent manner as demonstrated by the results shown in Figure 13 . At 4 hours, approximately 65% of the non-conjugated parental anti-5T4 antibody or ADC was internalized (range was a high of 70% for L398C to a low of 54% for L443C).
  • the ADC platform was also tested to determine whether it can mediate a cytotoxic effect on the target cells while not significantly affecting non-target cells. That is, the ADC, whilst carrying a cytotoxic payload, must still specifically bind to target cells while not significantly binding to non-target cells, then it must internalize and deliver the payload to a compartment where it will then mediate a cytotoxic effect to the target cells while sparing non-target cells which may be in close proximity.
  • novel ADCs of the present disclosure were subjected to this test and, as shown below, were able to bind to target cells while carrying a true linker and payload (not just the non-toxic vitamin biotin), be internalized, and mediate a cytotoxic effect to target cells, while not affecting non-target cells. This effect was comparable to the parental antibody comprising a wild type-IgG1 Fc region.
  • the vcMMAD conjugated Cys mutant Abs were approximately 10-fold more potent than the mcMMAD ADCs in inhibiting the growth in the 5T4+ cells.
  • the vcMMAD ADCs are linked with a more labile cathepsin sensitive vc linker and thus are more active in inhibiting cell growth than the more stably linked mcMMAD ADCs. Being more labile, the vc-linked ADCs also tend to be more toxic in animals. Both linker types have been tested in the clinic as ADCs.
  • novel cys mutants of the disclosure provide an effective platform for production of effective homogenous ADCs which can deliver a cytotoxic payload with precise stoichiometry of DAR and thereby provide a therapeutic effect.
  • the cytotoxcity observed for these ADCs is dependent upon antigen expression and antibody loading (DAR).
  • Table 21 illustrates the cytotoxicity of anti-Her2 mutants conjugated to the payload mcMMAD.
  • the data disclosed further demonstrate the cytotoxicity of anti-Her2 Fc mutants and Fc and kappa chain double-mutants conjugated to MMAD via mc, MalPeg6C2 and MalPeg3C2 linkers. Again, the increased loading of the double mutants is reflected by an increase in potency against Her2 expressing cell lines, BT474 and N87 (both considered to be high-expressers of Her2).
  • the ADCs were between 100 and 1000-fold less active against a non-Her2 expressing cell line (MDA-MB468).
  • the Fc region of IgG1 may mediate desirable effector functions, such as ADCC, which may provide additional therapeutic effects to the antibody. Accordingly, the effector function, e.g., ability to mediate ADCC, of the cys mutant antibodies of the present disclosure was assessed as follows.
  • the exposure values were approximately 58%, 61% and 55% higher in mice given 5T4-L398C-mcMMAD, 5T4-V422C-mcMMAD and 5T4-L443C-mcMMAD, respectively, compared to those dosed with the 5T4-mcMMAD (conventional cysteine conjugation) ADC as shown in Table 23.
  • the ADC exposure of 5T4-E380C-mcMMAD was lower (-9%) than the conventional ADC (5T4-mcMMAD).
  • cysteine-mutant ADCs e.g. 5T4-L443C-mcMMAD
  • ADCs e.g. 5T4-L443C-mcMMAD
  • administration of ADCs produced using the novel site specific conjugation methodology via particular engineered cysteine positions of this disclosure can result in more efficient delivery of the cytotoxic payload to the target tumor site compared to conventional ADCs.
  • Sites to engineer reactive cysteines were selected in the Kappa light chain constant region to expand diversity of positions for site-specific conjugation and to enable conjugation of 4 toxic payloads per antibody by combining engineered Kappa regions with select single Fc-region cysteine mutants.
  • Preferred positions for engineered cysteines in the Kappa constant region have predicted pKa values of 9.5-11.5 and predicted side chain solvent accessibility of 15-60 ⁇ 2 , properties which are predicted to mimic the most successful conjugated cysteine mutants disclosed previously herein, including, but not limited to Q347C, E380C, K392C, and L443C.
  • Table 24 sets forth the location of the mutations relative to wild type endogenous human Kappa constant region wherein the amino acid residue was mutated to cysteine for thiol reactive site-specific conjugation. Table 24 indicates the positions where human Kappa residues were replaced with reactive cysteines. Positions were defined by the Kabat numbering system as set forth in Kabat et al. (1991, NIH Publication 91 - 3242, National Technical Information Service, Springfield, VA), so all positions are numbered according to the Kabat system.
  • Human Kappa constant regions comprising engineered single cysteines at these novel positions shown in Table 24 were incorporated into an anti-Her2 antibody (amino acid sequences of the VH and VL domains of an exemplary Her2 antibody are show in Figures 17C and 17D , respectively) for further evaluation.
  • the nucleic acid encoding the anti-Her2 antibody human wild type Kappa constant region was removed from the expression vector by restriction enzyme digestion and replaced with a nucleic acid encoding human light chain constant Kappa regions comprising the single engineered cysteine residues using T4 DNA Ligase (New England Biolabs Inc., Ipswich, Massachusetts). The resulting nucleic acid was sequence confirmed for each construct.
  • HMW high molecular weight
  • maleimides can be transferred both in vitro and in vivo to exogenous thiol nucleophiles (see, e.g., Shen et al., 2012, Nature Biotech. 30(2):184-185 ).
  • GSH aqueous glutathione
  • a novel assay was developed that involves the treatment of the maleimide-linked ADC with excess aqueous glutathione (GSH) or plasma. Aliquots of the reaction mixture are analyzed at various timepoints to determine the loading of ADCs.
  • the ADC sample (30 ⁇ g) in PBS was mixed with glutathione (GSH) solution to produce final concentration of GSH of 0.5 mM and 3 mg/mL protein concentration.
  • GSH glutathione
  • a control sample was likewise prepared from 30 ⁇ g ADC diluted to 3 mg/mL in PBS.
  • the GSH-treated ADC sample and the control ADC sample were incubated at 37°C and were sampled at 0, 3, and 6 days. Aliquots were reduced with excess TCEP, acidified by adding 0.1% formic acid solution with 10% acetonitrile and analyzed by for loading by LC/MS as described below.
  • Sample analysis Analysis was performed using an Agilent 1100 capillary HPLC coupled with Waters Xevo G2 Q-TOF mass spectrometer. The analytes were loaded onto a Zorbax Poroshell 300SB C8 column (0.5 mm X 75 mm, maintained at 80°C) with 0.1% formic acid, and eluted using a gradient of 20-40% buffer B (80% acetonitrile, 18% 1-propanol, 2% water with 0.1% formic acid) at a flow rate of 20 ⁇ l/min over 5.5 minutes. Mass spectrometric detection was carried out in positive, sensitivity mode with capillary voltage set at 3.3 kV.
  • the engineered reactive cysteines selected in the Lambda light chain constant region disclosed herein expand diversity of positions for site-specific conjugation and enable conjugation of 4 toxic payloads per antibody by combining engineered Lambda regions with select single Fc-region cysteine mutants.
  • Preferred positions for engineered cysteines in the Lambda constant region of the disclosure have predicted pKa values of 9.5-11.5 and predicted side chain solvent accessibility of 15-60 ⁇ 2 .
  • these properties are shared with the preferred conjugated cysteine mutants disclosed previously herein, including, but not limited to, the heavy chain constant domain cysteines engineered at the following positions: Q347C, E380C, K392C, and L443C.
  • Each position was examined in each crystal structure by first mutating the position to cysteine and predicting the rotamer with SCWRL4 ( Krivov et al., 2009, Proteins 77(4):778-795 ), then by predicting the cysteine side chain pKa (using methods such as those described in, inter alia, Spassov and Yan, 2008, Protein Sci. 17:1955-1970 ) and side chain solvent accessibility using Discovery Studio 3.0 (Accelrys, Inc., San Diego, CA). Table 27 sets forth the location of the mutations relative to wild type endogenous human Lambda constant region wherein the amino acid residue was mutated to cysteine for thiol reactive site-specific conjugation.
  • Table 27 indicates the positions where human Lambda residues were replaced with reactive cysteines. Positions were defined by the Lambda numbering system as set forth in Kabat et al. (1991, NIH Publication 91 - 3242, National Technical Information Service, Springfield, VA), such that all positions are numbered according to the Kabat system.
  • the in vivo PK parameters of various site-specific conjugated ADCs of the disclosure were assessed in a mouse model. Briefly, the PK of various site-specific anti-Her2 conjugated ADCs loaded with mcMMAD using a MalPeg6C2_Aur linker-payload, where "Aur” is a proprietary auristatin payload also referred to as "8261" and disclosed in International Patent Application No. PCT/IB2012/056224 filed November 7, 2012 , were determined and the results are shown in Figure 21 .
  • DAR 4)
  • Both types of site specific ADCs had significant improvement in ADC/antibody ratios.
  • anti-Her2-mcMMAD PK data correlated with the PK parameters determined for comparable ADCs on an anti-5T4 antibody, suggesting that the engineered cysteine positions can be used across multiple antibody platforms to generate stable conjugates.
  • Rat toxicology studies were performed using anti-Her2-L443C-vc0101, anti-Her2-MaIPeg6C2-MMAD and anti-Her2-MalPeg6C2-Aur conjugates in the N87 gastric carcinoma model.
  • One site-specific conjugate of the disclosure, L443C-vc0101 demonstrated a better toxicity profile at the highest payload dose tested than the conventionally conjugated Her2-vc0101. Similar, but slightly less pronounced improvement in safety relative to the conventional conjugate was also observed for Her2-L443C-MalPeg6C2-Aur site-specific ADC.
  • TI values were determined by using the ratio of cNOAEL (statistically derived N o O bserved A dverse E ffect L evels based on the continuous response variable) from rat toxicology studies to efficacy defined as Tumor Static Concentration (TSC).
  • TSC Tumor Static Concentration

Claims (15)

  1. Gentechnisch manipuliertes humanes IgG-Polypeptid der konstanten Domäne (Cγ) der schweren Kette oder Teil davon, umfassend wenigstens eine Aminosäuresubstitution, zur Einführung eines Cysteinrests, der für die Konjugation nützlich ist, wobei sich die wenigstens eine Substitution an K246 gemäß dem EU-Indexnummerierungssystem, wie im EU-Index von Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85 dargelegt ist, befindet.
  2. Gentechnisch manipuliertes Cy-Polypeptid oder Teil davon, wie in Anspruch 1 beansprucht, umfassend wenigstens eine weitere Aminosäuresubstitution, ausgewählt aus der Gruppe, bestehend aus D249, D265, S267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, E318, K320, I332, E333, K334, I336, E345, Q347, S354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, N421, M428, A431, L432, T437, Q438, K439, L443 und S444, gemäß den EU-Index von Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1) :78-85.
  3. Gentechnisch manipuliertes Cy-Polypeptid gemäß Anspruch 1, wobei das Polypeptid ferner wenigstens eine Mutation umfasst, ausgewählt aus der Gruppe, bestehend aus einer Mutation an Aminosäureposition 284, 287, A327, N384, L398 und V422, gemäß dem EU-Index von Edelman at al., 1969, Proc. Natl. Acad. Sci. USA 63(1) :78-85.
  4. Gentechnisch manipuliertes Cy-Polypeptid gemäß Anspruch 1, wobei das Polypeptid SEQ ID NO:108 umfasst.
  5. Gentechnisch manipuliertes Cy-Polypeptid gemäß Anspruch 4, wobei das gentechnisch manipulierte Cy-Polypeptid SEQ ID NO:6 umfasst.
  6. Gentechnisch manipuliertes Cy-Polypeptid oder ein Teil davon gemäß Anspruch 1, wobei das gentechnisch manipulierte Polypeptid an ein oder mehrere eines zytotoxischen Mittels, eines zytostatischen Mittels, eines chemotherapeutischen Mittels, eines Toxins, eines Radionuklids, einer DNA, einer RNA, einer siRNA, einer microRNA, einer Peptidnucleinsäure, einer nichtnatürlichen Aminosäure, eines Peptids, eines Enzyms, einer Fluoreszenzmarkierung und eine Biotins, wobei sich die Konjugation am substituierten Cystein befindet und wobei das zytotoxische Mittel gegebenenfalls an das Polypeptid über einen Linker konjugiert ist und der Linker ausgewählt sein kann aus der Gruppe, bestehend aus mc (Maleimidocaproyl), val-cit (Valin-Citrullin), mc-val-cit (Maleimidocaproyl-Valin-Citrullin), mc-val-cit-PABC (Maleimidocaproyl-Valin-Citrullin-p-Aminobenzylcarbamat), Mal-PEG2C2 (Maleimido-[CH2CH2O]2CH2CH2C(=O)), Mal-PEG3C2 (Maleimido-[CH2CH2O]3CH2CH2C(=O)) und Mal-PEG6C2 (Maleimido-[CH2CH2O]6CH2CH2C(=O)).
  7. Gentechnisch manipuliertes Cy-Polypeptid oder ein Teil davon gemäß Anspruch 6, wobei das zytotoxische Mittel ausgewählt ist aus der Gruppe, bestehend aus einem Auristatin, einem Maytansinoid und einem Calicheamicin.
  8. Gentechnisch manipuliertes Cy-Polypeptid oder ein Teil davon gemäß Anspruch 7, wobei der Linker und das zytotoxische Mittel ausgewählt sind aus der Gruppe, bestehend aus Maleimidocaproyl-Monomethyl-Auristatin-D (mcMMAD), Maleimidocaproyl-0101 (mc0101), Maleimidocaproyl-3377 (mc3377), Maleimidocaproyl-8261 (mc8261), Valin-Citrullin-Monomethyl-Auristatin-D (vcMMAD), Valin-Citrullin-0101 (vc0101), Valin-Citrullin-3377 (vc3377), Valin-Citrullin-8261 (vc8261), mcValCitPABCMMAD (Maleimidocaproyl-Valin-Citrullin-Monomethyl-Auristatin-D), mcValCit0101 (Maleimidocaproyl-Valin-Citrullin-0101), mcValCit3377 (Maleimidocaproyl-Valin-Citrullin-3377), mcValCit8261 (Maleimidocaproyl-Valin-Citrullin-8261), Mal-PEG2C2-MMAD, Mal-PEG3C2-MMAD, Mal-PEG6C2-MMAD, Mal-PEG2C2-0101, Mal-PEG3C-0101, Mal-PEG6C2-0101, Mal-PEG2C2-3377, Mal-PEG3C2-3377 und Mal-PEG6C2-3377, Mal-PEG2C2-8261, Mal-PEG3C2-8261 und Mal-PEG6C2-8261.
  9. Fc-Fusionsprotein, umfassend das gentechnisch manipulierte Cy-Polypeptid gemäß einem der vorangehenden Ansprüche.
  10. Antikörper oder Antigen-bindender Teil davon, umfassend das gentechnisch manipulierte Cy-Polypeptid gemäß einem der Ansprüche 1 bis 9 und ferner umfassend eine leichte Kette, umfassend eine gentechnisch manipulierte konstante Domäne, ausgewählt aus der Gruppe, bestehend aus:
    (a) einem gentechnisch manipulierten humanen Polypeptid der konstanten Domäne (Cλ) der leichten lambda-Kette oder einem Teil davon, umfassend wenigstens eine Aminosäuresubstitution, ausgewählt aus der Gruppe, bestehend aus K110, A111, L125, K149C, V155, G158, T161, Q185, S188, H189, S191, T197, V205, E206, K207, T208 und A210, gemäß der Kabat-Nummerierung; und
    (b) einem gentechnisch manipulierten humanen Polypeptid der konstanten Domäne (CK) der leichten kappa-Kette oder einem Teil davon, umfassend wenigstens eine Aminosäuresubstitution, ausgewählt aus der Gruppe, bestehend aus A111, K183 und N210, gemäß der Kabat-Nummerierung.
  11. Pharmazeutische Zusammensetzung, umfassend das gentechnisch manipulierte Cy-Polypeptid oder einen Teil davon gemäß einem der Ansprüche 1-9 oder einen Antikörper oder Antigen-bindenden Teil davon, wie in Anspruch 10 beansprucht, und einen pharmazeutisch verträglichen Träger.
  12. Zusammensetzung, wie in Anspruch 11 beansprucht, zur Verwendung in einem Verfahren zur Behandlung von Krebs, Autoimmun-, Entzündungs- oder Infektionskrankheiten und -störungen in einem Subjekt, das diese benötigt.
  13. Nucleinsäure, die das gentechnisch manipulierte Cy-Polypeptid oder einen Teil davon gemäß den Ansprüchen 1-9 oder den Antikörper oder Antigen-bindenden Teil davon, wie in Anspruch 10 beansprucht, codiert.
  14. Wirtszelle, umfassend die Nucleinsäure gemäß Anspruch 13.
  15. Verfahren zur Herstellung eines gentechnisch manipulierten Cy-Polypeptids oder eines Teils davon oder eines Antikörpers oder Antigen-bindenden Teils davon, umfassend das Inkubieren der Wirtszelle gemäß Anspruch 14 bei Bedingungen, die zur Expression des gentechnisch manipulierten Cy-Polypeptids oder eines Teils davon oder des Antikörpers oder Antigen-bindenden Teils davon geeignet sind, und Isolieren des gentechnisch manipulierten Cy-Polypeptids oder eines Teils davon oder des Antikörpers oder Antigen-bindenden Teils davon.
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